Preparation of (r,r)-fenoterol and (r,r)-or (r,s)-fenoterol analogues and their use in treating congestive heart failure

ABSTRACT

This disclosure concerns the discovery of (R,R)- and (R,S)-fenoterol analogues which are highly effective at binding β2-adrenergic receptors. Exemplary chemical structures for these analogues are provided. Also provided are pharmaceutical compositions including the disclosed (R,R)-fenoterol and fenoterol analogues, and methods of using such compounds and compositions for the treatment of cardiac disorders such as congestive heart failure and pulmonary disorders such as asthma or chronic obstructive pulmonary disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/333,866, filed Dec. 21, 2011, which is a continuation of U.S. patentapplication Ser. No. 12/376,945, filed Feb. 9, 2009, now abandoned,which is a §371 U.S. National Stage of International Application No.PCT/US2007/075731, filed Aug. 10, 2007, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Patent Application No. 60/837,161, filed Aug. 10, 2006 andU.S. Provisional Application No. 60/927,825, filed May 3, 2007, all ofwhich are incorporated herein in their entirety by reference.

FIELD

The present disclosure relates to the field of pharmaceuticalcompositions and in particular, to the preparation of (R,R)-fenoteroland (R,R)- or (R,S)-fenoterol analogues and their use in treatingcongestive heart failure.

BACKGROUND

Fenoterol,5-[1-hydroxy-2[[2-(4-hydroxyphenyl)-1-methylethyl]-amino]ethyl]1,2-benzenediol,is a β2-adrenergic receptor agonist that has traditionally been used forthe treatment of pulmonary disorders such as asthma. This drug has twochiral (asymmetric) carbons that can each be independently arranged inan R or S configuration, so that the drug exists in distinct (R,R),(R,S), (S,R) and (S,S) forms known as stereoisomers. Fenoterol iscommercially available as a racemic mixture of the (R,R)- and(S,S)-compounds.

Fenoterol is known to act as an agonist that binds to and activates theβ2-adrenergic receptor. This activity has led to its clinical use in thetreatment of asthma because this agonist's activity dilates constrictedairways. Additional therapeutic uses for fenoterol remain to bethoroughly explored. Pharmacological studies of the class of drugs thatincludes fenoterol have shown that only one of the enantiomers isresponsible for generating brochodilation. For example, studies havedemonstrated that the primary bronchodilatory activity for racemic(±)-fenoterol resides in the (R,R)-isomer of fenoterol. Further, it hasrecently become apparent that the inactive enantiomer may be associatedwith adverse effects. For instance, the diastereomer (S,S)-fenoterol hasbeen demonstrated to cause adverse side effects or development oftolerance often associated with β2-adrenergic receptor agonisttreatment.

It would therefore be advantageous to provide fenoterol compositionsthat were effective at treating disorders such as asthma, chronicobstructive pulmonary disease, or congestive heart failure, but hadreduced side effects such as hypersensitivity and drug resistance(tolerance).

SUMMARY

This disclosure concerns the discovery of fenoterol analogues that arehighly effective at binding β2-adrenergic receptors. Exemplary chemicalstructures for these analogues are provided throughout the disclosure.By way of example, such compounds are represented by the followinggeneral formula:

wherein R₁-R₃ independently are hydrogen, acyl, alkoxy carbonyl, aminocarbonyl (carbamoyl) or a combination thereof;

R₄ is H or lower alkyl;

R₅ is lower alkyl,

wherein X, Y¹, Y² and Y³ independently are hydrogen, —OR₆ and —NR₇R₈;

R₆ is independently hydrogen, lower alkyl, acyl, alkoxy carbonyl oramino carbonyl; and

R₇ and R₈ independently are hydrogen, lower alkyl, alkoxy carbonyl, acylor amino carbonyl.

Pharmaceutical compositions containing, and methods of using(R,R)-fenoterol and fenoterol analogues are also provided. For example,the disclosed (R,R)-fenoterol and (R,R)- or (R,S)-fenoterol analogues(e.g., (R,R)-methoxyfenoterol, (R,R)-napthylfenoterol, and(R,S)-napthylfenoterol) are effective at treating cardiac disorders orpulmonary disorders.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the chromatographic separation of (S,S)- and(R,R)-fenoterol.

FIG. 2A provides an ultraviolet spectra of (R,R)- and (S,S)-fenoterol.

FIG. 2B illustrates a circular dichroism spectra of (R,R)- and(S,S)-fenoterol.

FIG. 3 provides frontal chromatographic elution profiles of[⁺H]-(±)-fenoterol produced by the addition of (R,R)-fenoterol to therunning buffer.

FIG. 4A is a graph including dose-response curves generated by treatingfreshly isolated rat ventricular myocytes with (±)-fenoterol,(R,R)-fenoterol or (S,S)-fenoterol.

FIG. 4B is a graph including dose-response curves of T₅₀% relaxation infreshly isolated rat ventricular myocytes generated by treatment with(±)-fenoterol, (R,R)-fenoterol or (S,S)-fenoterol.

FIG. 5 illustrates the chemical structures of the stereoisomers offenoterol and fenoterol analogs (compounds 2-7).

FIG. 6 illustrates the chemical structures of compounds 47-51.

FIG. 7 illustrates the effect of fenoterol and fenoterol analogs on cellcontraction in single ventricular myocytes.

FIG. 8 is a graph illustrating time-dependent mean plasma concentrationof (R,R)-fenoterol, (R,R)-methoxyfenoterol and (R,S)-napthylfenoterolafter administration (5 mg/mL).

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS I. Introduction

This disclosure provides fenoterol analogues that bind the β2-adrenergicreceptor with comparable or greater activity than fenoterol. In oneembodiment, the optically active fenoterol analogues are substantiallypurified from a racemic mixture. For example, an optically activefenoterol analogue is purified to represent greater than 90%, oftengreater than 95% of the composition. These analogues can be used totreat pulmonary disorders such as asthma and chronic obstructivepulmonary disease that have previously been treated with (±)-fenoterol.Use of the disclosed fenoterol analogues with an equal to higherefficacy than (±)-fenoterol can possibly reduce adverse effectspreviously observed with (±)-fenoterol. For example, use of a lowerconcentration of the fenoterol analogues to obtain a therapeuticeffective response is expected to reduce side-effects such ashypersensitivity and drug resistance (tolerance) observed with thecommercially available (±)-fenoterol. Also, purification of theanalogues removes contaminants such as the inactive enantiomer which canbe responsible for these adverse effects.

The present disclosure also demonstrates that (R,R)-fenoterol is theactive component of commercially available (±)-fenoterol. It isspecifically contemplated that (R,R)-fenoterol as well as disclosed(R,R)- and (R,S)-fenoterol analogues can be used to treat cardiacdisorders such as congestive heart failure. Use of substantiallyoptically pure (R,R)-fenoterol or (R,R)- or (R,S)-fenoterol analogues totreat congestive heart failure is expected to reduce the incidence ofside-effects caused by physiologically less active forms of the drug.

II. Abbreviations and Terms

-   -   AR: adrenergic receptor    -   CD: circular dichroism    -   CoMFA: comparative molecular field analysis    -   HPLC: high performance liquid chromatography    -   IAM-PC: immobilized artificial membrane chromatographic support    -   ICYP: [¹²⁵I]cyanopindolol    -   UV: ultraviolet

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the disclosed subject matter belongs.Definitions of common terms in chemistry terms may be found in TheMcGraw-Hill Dictionary of Chemical Terms, 1985, and The CondensedChemical Dictionary, 1981. As used herein, the singular terms “a,” “an,”and “the” include plural referents unless context clearly indicatesotherwise. Similarly, the word “or” is intended to include “and” unlessthe context clearly indicates otherwise. Also, as used herein, the term“comprises” means “includes.” Hence “comprising A or B” means includingA, B, or A and B.

Except as otherwise noted, any quantitative values are approximatewhether the word “about” or “approximately” or the like are stated ornot. The materials, methods, and examples described herein areillustrative only and not intended to be limiting. Any molecular weightor molecular mass values are approximate and are provided only fordescription. Except as otherwise noted, the methods and techniques ofthe present invention are generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See, e.g., Loudon, Organic Chemistry, FourthEdition, New York: Oxford University Press, 2002, pp. 360-361,1084-1085; Smith and March, March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience,2001; or Vogel, A Textbook of Practical Organic Chemistry, IncludingQualitative Organic Analysis, Fourth Edition, New York: Longman, 1978.

In order to facilitate review of the various embodiments disclosedherein, the following explanations of specific terms are provided:

Acyl: A group of the formula RC(O)— wherein R is an organic group.

Acyloxy: A group having the structure —OC(O)R, where R may be anoptionally substituted alkyl or optionally substituted aryl. “Loweracyloxy” groups are those where R contains from 1 to 10 (such as from 1to 6) carbon atoms.

Alkoxy: A radical (or substituent) having the structure —O—R, where R isa substituted or unsubstituted alkyl. Methoxy (—OCH₃) is an exemplaryalkoxy group. In a substituted alkoxy, R is alkyl substituted with anon-interfering substituent. “Thioalkoxy” refers to —S—R, where R issubstituted or unsubstituted alkyl. “Haloalkyloxy” means a radical —ORwhere R is a haloalkyl.

Alkoxy carbonyl: A group of the formula —C(O)OR, where R may be anoptionally substituted alkyl or optionally substituted aryl. “Loweralkoxy carbonyl” groups are those where R contains from 1 to 10 (such asfrom 1 to 6) carbon atoms.

Alkyl: An acyclic, saturated, branched- or straight-chain hydrocarbonradical, which, unless expressly stated otherwise, contains from one tofifteen carbon atoms; for example, from one to ten, from one to six, orfrom one to four carbon atoms. This term includes, for example, groupssuch as methyl, ethyl, n-propyl, isopropyl, isobutyl, t-butyl, pentyl,heptyl, octyl, nonyl, decyl, or dodecyl. The term “lower alkyl” refersto an alkyl group containing from one to ten carbon atoms. Unlessexpressly referred to as an “unsubstituted alkyl,” alkyl groups caneither be unsubstituted or substituted. An alkyl group can besubstituted with one or more substituents (for example, up to twosubstituents for each methylene carbon in an alkyl chain). Exemplaryalkyl substituents include, for instance, amino groups, amide,sulfonamide, halogen, cyano, carboxy, hydroxy, mercapto,trifluoromethyl, alkyl, alkoxy (such as methoxy), alkylthio, thioalkoxy,arylalkyl, heteroaryl, alkylamino, dialkylamino, alkylsulfano, keto, orother functionality.

Amino carbonyl (carbamoyl): A group of the formula —OCN(R)R′—, wherein Rand R′ are independently of each other hydrogen or a lower alkyl group.

Asthma: Asthma is a disease of the respiratory system in which theairways constrict, become inflamed, and are lined with excessive amountsof mucus, often in response to one or more “triggers,” such as exposureto an environmental stimulant (or allergen), cold air, exercise, oremotional stress. This airway narrowing causes symptoms such aswheezing, shortness of breath, chest tightness, and coughing. Thedisorder is a chronic or recurring inflammatory condition in which theairways develop increased responsiveness to the various stimuli,characterized by bronchial hyper-responsiveness, inflammation, increasedmucus production, and intermittent airway obstruction.

Carbamate: A group of the formula —OC(O)N(R)—, wherein R is H, or analiphatic group, such as a lower alkyl group or an aralkyl group

Cardiac Disorder or Disease: In general, a cardiac disorder/disease is aclass of disorders/diseases that involve the heart and/or blood vessels(arteries and veins). In a particular example, cardiac disorder/diseaseincludes congestive heart failure.

Chronic Obstructive Pulmonary Disease: A group of respiratory tractdiseases including chronic bronchitis, emphysema and bronchiectasis thatare characterized by airflow obstruction or limitation that is not fullyreversible. The airflow limitation is usually progressive and associatedwith an abnormal inflammatory response of the lungs to noxious particlesor gases.

Congestive Heart Failure: Heart failure in which the heart is unable tomaintain an adequate circulation of blood in the bodily tissues or topump out the venous blood returned to it by the veins.

Derivative: A chemical substance that differs from another chemicalsubstance by one or more functional groups. Preferably, a derivative(such as a fenoterol analogue) retains a biological activity (such asβ2-adrenergic receptor stimulation) of a molecule from which it wasderived (such as a fenoterol).

Isomers: Compounds that have the same molecular formula but differ inthe nature or sequence of bonding of their atoms or the arrangement oftheir atoms in space are termed “isomers”. Isomers that differ in thearrangement of their atoms in space are termed “stereoisomers”.Stereoisomers that are not mirror images of one another are termed“diastereomers” and those that are non-superimposable mirror images ofeach other are termed “enantiomers.” When a compound has an asymmetriccenter, for example, if a carbon atom is bonded to four differentgroups, a pair of enantiomers is possible. An enantiomer can becharacterized by the absolute configuration of its asymmetric center andis described by the R- and S-sequencing rules of Cahn and Prelog, or bythe manner in which the molecule rotates the plane of polarized lightand designated as dextrorotatory or levorotatory (i.e., as (+) or (−)isomers, respectively). A chiral compound can exist as either anindividual enantiomer or as a mixture thereof. A mixture containingequal proportions of the enantiomers is called a “racemic mixture.”

The compounds described herein may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof. Unless indicated otherwise,the description or naming of a particular compound in the specificationand claims is intended to include both individual enantiomers andmixtures, racemic or otherwise, thereof. The methods for thedetermination of stereochemistry and the separation of stereoisomers arewell-known in the art (see, e.g., March, Advanced Organic Chemistry, 4thedition, New York: John Wiley and Sons, 1992, Chapter 4).

Optional: “Optional” or “optionally” means that the subsequentlydescribed event or circumstance can but need not occur, and that thedescription includes instances where said event or circumstance occursand instances where it does not.

Pharmaceutically Acceptable Carriers: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 19th Edition (1995), describes compositions andformulations suitable for pharmaceutical delivery of one or moretherapeutic compounds or molecules, such as one or more nucleic acidmolecules, proteins or antibodies that bind these proteins, andadditional pharmaceutical agents.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Phenyl: Phenyl groups may be unsubstituted or substituted with one, twoor three substituents, with substituent(s) independently selected fromalkyl, heteroalkyl, aliphatic, heteroaliphatic, thioalkoxy, halo,haloalkyl (such as —CF₃), nitro, cyano, —OR (where R is hydrogen oralkyl), —N(R)R′ (where R and R′ are independently of each other hydrogenor alkyl), —COOR (where R is hydrogen or alkyl) or —C(O)N(R′)R″ (whereR′ and R″ are independently selected from hydrogen or alkyl).

Pulmonary Disorder or Disease: In general, pulmonary disorder/diseaseincludes any disorder/disease pertaining to the lungs. In a particularexample, pulmonary disorder/disease includes asthma and chronicobstructive pulmonary disease.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purifiedpreparation is one in which a desired component such as an(R,R)-enantiomer of fenoterol is more enriched than it was in apreceding environment such as in a (±)-fenoterol mixture. A desiredcomponent such as (R,R)-enantiomer of fenoterol is considered to bepurified, for example, when at least about 70%, 80%, 85%, 90%, 92%, 95%,97%, 98%, or 99% of a sample by weight is composed of the desiredcomponent. Purity of a compound may be determined, for example, by highperformance liquid chromatography (HPLC) or other conventional methods.In an example, the specific fenoterol analogue enantiomers are purifiedto represent greater than 90%, often greater than 95% of the otherenantiomers present in a purified preparation. In other cases, thepurified preparation may be essentially homogeneous, wherein otherstereoisomers are less than 1%.

Compounds described herein may be obtained in a purified form orpurified by any of the means known in the art, including silica geland/or alumina chromatography. See, e.g., Introduction to Modern LiquidChromatography, 2nd Edition, ed. by Snyder and Kirkland, New York:JohnWiley and Sons, 1979; and Thin Layer Chromatography, ed. by Stahl, NewYork: Springer Verlag, 1969. In an example, a compound includes purifiedfenoterol or fenoterol analogue with a purity of at least about 70%,80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% of a sample by weight relativeto other contaminants. In a further example, a compound includes atleast two purified stereoisomers each with a purity of at least about70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% of a sample by weightrelative to other contaminants. For instance, a compound can include asubstantially purified (R,R)-fenoterol analogue and a substantiallypurified (R,S)-fenoterol analogue.

Subject: The term “subject” includes both human and veterinary subjects,for example, humans, non-human primates, dogs, cats, horses, rats, mice,and cows. Similarly, the term mammal includes both human and non-humanmammals.

Treating or treatment: With respect to disease, either term includes (1)preventing the disease, e.g., causing the clinical symptoms of thedisease not to develop in a subject that may be exposed to orpredisposed to the disease but does not yet experience or displaysymptoms of the disease, (2) inhibiting the disease, e.g., arresting thedevelopment of the disease or its clinical symptoms, or (3) relievingthe disease, e.g., causing regression of the disease or its clinicalsymptoms.

Therapeutically Effective Amount: A quantity of a specified agentsufficient to achieve a desired effect in a subject being treated withthat agent. For example, this may be the amount of (R,R)-fenoterol or(R,R)- or (R,S)-fenoterol analogue useful in preventing, reducing,and/or inhibiting, and/or treating a cardiac disorder such as congestiveheart failure. Ideally, a therapeutically effective amount of an agentis an amount sufficient to prevent, reduce, and/or inhibit, and/or treatthe disorder in a subject without causing a substantial cytotoxic effectin the subject. The effective amount of an agent useful for preventing,reducing, and/or inhibiting, and/or treating a disorder in a subjectwill be dependent on the subject being treated, the severity of thedisorder, and the manner of administration of the therapeuticcomposition.

III. (R,R)-Fenoterol and Fenoterol Analogues

A. Chemical Structure

Some exemplary fenoterol analogues disclosed herein have the formula:

wherein R₁-R₃ independently are hydrogen, acyl, alkoxy carbonyl, aminocarbonyl or a combination thereof;

R₄ is H or lower alkyl;

R₅ is lower alkyl,

wherein X and Y independently are selected from hydrogen, lower —OR₆ and—NR₇R₈;

R₆ is lower alkyl or acyl; and

R₇ and R₈ independently are hydrogen, lower alkyl, alkoxy carbonyl, acylor amino carbonyl.

With continued reference to the general formula for fenoterol analoguesabove, Y may be —OH

In one embodiment, R₅ is a 1- or 2-napthyl derivative optionally having1, 2 or 3 substituents. Examples of such R₅ groups are represented bythe formula

wherein Y¹, Y² and Y³ independently are hydrogen, lower —OR₆ and —NR₇R₈;

R₆ is independently for each occurrence selected from lower alkyl andacyl; and

R₇ and R₈ independently are hydrogen, lower alkyl, alkoxy carbonyl, acylor amino carbonyl (carbamoyl). In particular compounds at least one ofY¹, Y² and Y³ is —OCH₃.

Particular R₅ groups include those represented by the formulas

wherein R₆ is lower alkyl, such as methyl, ethyl, propyl or isopropyl oracyl, such as acetyl.

Exemplary R₅ groups include

In one example, R₄ is lower alkyl and R₅ is

wherein X and Y independently are selected from H, lower alkyl —OR₆ and—NR₇R₈;

R₆ is lower alkyl; and

R₇ and R₈ independently are hydrogen or lower alkyl.

In a further example, R₄ is selected from ethyl, n-propyl, and isopropyland R₅ has the formula

wherein X is H, —OR₆ or —NR₇R₈. For example, R₆ may be methyl or R₇ andR₈ are hydrogen.

In an additional example, R₅ has the formula

In further embodiments, R₄ is selected from methyl, ethyl, n-propyl andisopropyl and R₅ represents

Examples of suitable groups for R₁-R₃ that can be cleaved in vivo toprovide a hydroxy group include, without limitation, acyl, acyloxy andalkoxy carbonyl groups. Compounds having such cleavable groups arereferred to as “prodrugs.” The term “prodrug,” as used herein, means acompound which includes a substituent that is convertible in vivo (e.g.,by hydrolysis) to a hydroxyl group. Various forms of prodrugs are knownin the art, for example, as discussed in Bundgaard, (ed.), Design ofProdrugs, Elsevier (1985); Widder, et al. (ed.), Methods in Enzymology,Vol. 4, Academic Press (1985); Krogsgaard-Larsen, et al., (ed), Designand Application of Prodrugs, Textbook of Drug Design and Development,Chapter 5, 113 191 (1991); Bundgaard, et al., Journal of Drug DeliveryReviews, 8:1 38(1992); Bundgaard, Pharmaceutical Sciences, 77:285 etseq. (1988); and Higuchi and Stella (eds.) Prodrugs as Novel DrugDelivery Systems, American Chemical Society (1975).

An exemplary (R,R)-compound has the chemical structure of:

X and R₁-R₃ are as described above.

An additional exemplary (R,R)-compound has the chemical structure:

An exemplary (R,S)-compound has the chemical structure:

wherein X and R₁-R₃ are as described above.

An additional exemplary (R,S)-compound has the chemical structure:

An exemplary (S,R)-compound has the chemical structure:

wherein X and R₁-R₃ are as described above.

An exemplary (S,S)-compound has the chemical structure:

wherein X and R₁-R₃ are as described above.

Examples of chemical structures illustrating the various stereoisomersof fenoterol are provided below.

Particular method embodiments contemplate the use of solvates (such ashydrates), pharmaceutically acceptable salts and/or different physicalforms of (R,R)-fenoterol or any of the fenoterol analogues hereindescribed.

1. Solvates, Salts and Physical Forms

“Solvate” means a physical association of a compound with one or moresolvent molecules. This physical association involves varying degrees ofionic and covalent bonding, including by way of example covalent adductsand hydrogen bonded solvates. In certain instances the solvate will becapable of isolation, for example when one or more solvent molecules areincorporated in the crystal lattice of the crystalline solid. “Solvate”encompasses both solution-phase and isolable solvates. Representativesolvates include ethanol associated compound, methanol associatedcompounds, and the like. “Hydrate” is a solvate wherein the solventmolecule(s) is/are H₂O.

The disclosed compounds also encompass salts including, if severalsalt-forming groups are present, mixed salts and/or internal salts. Thesalts are generally pharmaceutically-acceptable salts that arenon-toxic. Salts may be of any type (both organic and inorganic), suchas fumarates, hydrobromides, hydrochlorides, sulfates and phosphates. Inan example, salts include non-metals (e.g., halogens) that form groupVII in the periodic table of elements. For example, compounds may beprovided as a hydrobromide salt.

Additional examples of salt-forming groups include, but are not limitedto, a carboxyl group, a phosphonic acid group or a boronic acid group,that can form salts with suitable bases. These salts can include, forexample, nontoxic metal cations which are derived from metals of groupsIA, IB, IIA and IIB of the periodic table of the elements. In oneembodiment, alkali metal cations such as lithium, sodium or potassiumions, or alkaline earth metal cations such as magnesium or calcium ionscan be used. The salt can also be a zinc or an ammonium cation. The saltcan also be formed with suitable organic amines, such as unsubstitutedor hydroxyl-substituted mono-, di- or tri-alkylamines, in particularmono-, di- or tri-alkylamines, or with quaternary ammonium compounds,for example with N-methyl-N-ethylamine, diethylamine, triethylamine,mono-, bis- or tris-(2-hydroxy- lower alkyl)amines, such as mono-, bis-or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine ortris(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxy-loweralkyl)amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine ortri-(2-hydroxyethyl)amine, or N-methyl-D-glucamine, or quaternaryammonium compounds such as tetrabutylammonium salts.

Exemplary compounds disclosed herein possess at least one basic groupthat can form acid-base salts with inorganic acids. Examples of basicgroups include, but are not limited to, an amino group or imino group.Examples of inorganic acids that can form salts with such basic groupsinclude, but are not limited to, mineral acids such as hydrochloricacid, hydrobromic acid, sulfuric acid or phosphoric acid. Basic groupsalso can form salts with organic carboxylic acids, sulfonic acids, sulfoacids or phospho acids or N-substituted sulfamic acid, for exampleacetic acid, propionic acid, glycolic acid, succinic acid, maleic acid,hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid,tartaric acid, gluconic acid, glucaric acid, glucuronic acid, citricacid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid, and, in addition,with amino acids, for example with α-amino acids, and also withmethanesulfonic acid, ethanesulfonic acid, 2-hydroxymethanesulfonicacid, ethane-1,2-disulfonic acid, benzenedisulfonic acid,4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate or N-cyclohexylsulfamic acid(with formation of the cyclamates) or with other acidic organiccompounds, such as ascorbic acid. In a currently preferred embodiment,fenoterol is provided as a hydrobromide salt and exemplary fenoterolanalogues are provided as their fumarate salts.

Additional counterions for forming pharmaceutically acceptable salts arefound in Remington's Pharmaceutical Sciences, 19th Edition, MackPublishing Company, Easton, Pa., 1995. In one aspect, employing apharmaceutically acceptable salt may also serve to adjust the osmoticpressure of a composition.

In certain embodiments the compounds used in the method are provided arepolymorphous. As such, the compounds can be provided in two or morephysical forms, such as different crystal forms, crystalline, liquidcrystalline or non-crystalline (amorphous) forms.

2. Use for the Manufacture of a Medicament

Any of the above described compounds (e.g., (R,R)-fenoterol or fenoterolanalogues or a hydrate or pharmaceutically acceptable salt thereof) orcombinations thereof are intended for use in the manufacture of amedicament for β2-adrenergic receptor stimulation in a subject or forthe treatment of pulmonary and cardiac disorders (e.g., asthma andcongestive heart failure). Formulations suitable for such medicaments,subjects who may benefit from same and other related features aredescribed elsewhere herein.

B. Methods of Synthesis

The disclosed fenoterol analogues can be synthesized by any method knownin the art. Many general references providing commonly known chemicalsynthetic schemes and conditions useful for synthesizing the disclosedcompounds are available (see, e.g., Smith and March, March's AdvancedOrganic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978).

Compounds as described herein may be purified by any of the means knownin the art, including chromatographic means, such as HPLC, preparativethin layer chromatography, flash column chromatography and ion exchangechromatography. Any suitable stationary phase can be used, includingnormal and reversed phases as well as ionic resins. Most typically thedisclosed compounds are purified via open column chromatography or prepchromatography.

Suitable exemplary syntheses of fenoterol analogues are provided below:

Scheme III describes an exemplary synthesis for the chiral buildingblocks used in Scheme H. The (R)- and(S)-3′,5′-dibenzyloxyphenyl-bromohydrin enantiomers were obtained by theenantiospecific reduction of 3,5-dibenzyloxy-α-bromoacetophenone usingboron-methyl sulfide complex (BH₃SCH₃) and either (1R,2S)- or(1S,2R)-cis-1-amino-2-indanol. The required (R)- and(S)-2-benzylaminopropanes were prepared by enantioselectivecrystallization of the rac-2-benzylaminopropanes using either (R)- or(S)-mandelic acid as the counter ion.

IV. Pharmaceutical Compositions

The disclosed (R,R)-fenoterol and fenoterol analogues can be useful, atleast, for the treatment of pulmonary disorders such as asthma andchronic obstructive pulmonary disease (COPD) and cardiac disorders suchas congestive heart failure. Accordingly, pharmaceutical compositionscomprising at least one disclosed fenoterol compound or analogue arealso described herein.

Formulations for pharmaceutical compositions are well known in the art.For example, Remington's Pharmaceutical Sciences, by E. W. Martin, MackPublishing Co., Easton, Pa., 19th Edition, 1995, describes exemplaryformulations (and components thereof) suitable for pharmaceuticaldelivery of (R,R)-fenoterol and disclosed fenoterol analogues.Pharmaceutical compositions comprising at least one of these compoundscan be formulated for use in human or veterinary medicine. Particularformulations of a disclosed pharmaceutical composition may depend, forexample, on the mode of administration (e.g., oral or parenteral) and/oron the disorder to be treated (e.g., pulmonary disorder or cardiacdisorder such as congestive heart failure). In some embodiments,formulations include a pharmaceutically acceptable carrier in additionto at least one active ingredient, such as a fenoterol compound.

Pharmaceutically acceptable carriers useful for the disclosed methodsand compositions are conventional in the art. The nature of apharmaceutical carrier will depend on the particular mode ofadministration being employed. For example, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions such as powder, pill, tablet, or capsuleforms conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can optionally contain minor amounts ofnon-toxic auxiliary substances or excipients, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like;for example, sodium acetate or sorbitan monolaurate. Other non-limitingexcipients include, nonionic solubilizers, such as cremophor, orproteins, such as human serum albumin or plasma preparations.

The disclosed pharmaceutical compositions may be formulated as apharmaceutically acceptable salt. Pharmaceutically acceptable salts arenon-toxic salts of a free base form of a compound that possesses thedesired pharmacological activity of the free base. These salts may bederived from inorganic or organic acids. Non-limiting examples ofsuitable inorganic acids are hydrochloric acid, nitric acid, hydrobromicacid, sulfuric acid, hydriodic acid, and phosphoric acid. Non-limitingexamples of suitable organic acids are acetic acid, propionic acid,glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, methyl sulfonic acid,salicylic acid, formic acid, trichloroacetic acid, trifluoroacetic acid,gluconic acid, asparagic acid, aspartic acid, benzenesulfonic acid,p-toluenesulfonic acid, naphthalenesulfonic acid, and the like. Lists ofother suitable pharmaceutically acceptable salts are found inRemington's Pharmaceutical Sciences, 19th Edition, Mack PublishingCompany, Easton, Pa., 1995. A pharmaceutically acceptable salt may alsoserve to adjust the osmotic pressure of the composition.

The dosage form of a disclosed pharmaceutical composition will bedetermined by the mode of administration chosen. For example, inaddition to injectable fluids, oral dosage forms may be employed. Oralformulations may be liquid such as syrups, solutions or suspensions orsolid such as powders, pills, tablets, or capsules. Methods of preparingsuch dosage forms are known, or will be apparent, to those skilled inthe art.

Certain embodiments of the pharmaceutical compositions comprising adisclosed compound may be formulated in unit dosage form suitable forindividual administration of precise dosages. The amount of activeingredient such as (R,R)-fenoterol administered will depend on thesubject being treated, the severity of the disorder, and the manner ofadministration, and is known to those skilled in the art. Within thesebounds, the formulation to be administered will contain a quantity ofthe extracts or compounds disclosed herein in an amount effective toachieve the desired effect in the subject being treated. In particularexamples, for oral administration the compositions are provided in theform of a tablet containing from about 1.0 to about 50 mg of the activeingredient, particularly about 2.0 mg, about 2.5 mg, 5 mg, about 10 mg,or about 50 mg of the active ingredient for the symptomatic adjustmentof the dosage to the subject being treated. In one exemplary oral dosageregimen, a tablet containing from about 1 mg to about 50 mg (such asabout 2 mg to about 10 mg) active ingredient is administered two to fourtimes a day, such as two times, three times or four times.

V. Methods of Use

The present disclosure includes methods of treating disorders includingpulmonary and cardiac disorders. In some examples, the pulmonarydisorder is asthma or chronic obstructive pulmonary disease. In otherexamples, the cardiac disorder is congestive heart failure.

Disclosed methods includes administering (R,R)-fenoterol or a disclosedfenoterol analogue (and, optionally, one or more other pharmaceuticalagents) to a subject in a pharmaceutically acceptable carrier and in anamount effective to treat the pulmonary and/or cardiac disorder. Routesof administration useful in the disclosed methods include but are notlimited to oral and parenteral routes, such as intravenous (iv),intraperitoneal (ip), rectal, topical, ophthalmic, nasal, andtransdermal. Formulations for these dosage forms are described above.

An effective amount of (R,R)-fenoterol or a disclosed fenoterol analoguewill depend, at least, on the particular method of use, the subjectbeing treated, the severity of the disorder, and the manner ofadministration of the therapeutic composition. A “therapeuticallyeffective amount” of a composition is a quantity of a specified compoundsufficient to achieve a desired effect in a subject being treated. Forexample, this may be the amount of a (R,R)-fenoterol necessary toprevent, inhibit, reduce or relieve the pulmonary and/or cardiacdisorder and/or one or more symptoms of disorder in a subject. Ideally,a therapeutically effective amount of (R,R)-fenoterol or a disclosedfenoterol analogue is an amount sufficient to prevent, inhibit, reduceor relieve the pulmonary and/or cardiac disorder and/or one or moresymptoms of the disorder without causing a substantial cytotoxic effecton host cells.

Therapeutically effective doses of a disclosed fenoterol compound orpharmaceutical composition can be determined by one of skill in the art,with a goal of achieving concentrations that are at least as high as theEC₅₀ of the applicable compound disclosed in the examples herein. Anexample of a dosage range is from about 0.001 to about 10 mg/kg bodyweight orally in single or divided doses. In particular examples, adosage range is from about 0.005 to about 5 mg/kg body weight orally insingle or divided doses (assuming an average body weight ofapproximately 70 kg; values adjusted accordingly for persons weighingmore or less than average). For oral administration, the compositionsare, for example, provided in the form of a tablet containing from about1.0 to about 50 mg of the active ingredient, particularly about 2.5 mg,about 5 mg, about 10 mg, or about 50 mg of the active ingredient for thesymptomatic adjustment of the dosage to the subject being treated. Inone exemplary oral dosage regimen, a tablet containing from about 1 mgto about 50 mg active ingredient is administered two to four times aday, such as two times, three times or four times.

The specific dose level and frequency of dosage for any particularsubject may be varied and will depend upon a variety of factors,including the activity of the specific compound, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex and diet of the subject, mode and time of administration,rate of excretion, drug combination, and severity of the condition ofthe subject undergoing therapy.

The subject matter of the present disclosure is further illustrated bythe following non-limiting Examples.

EXAMPLES Example 1 Materials and Methods

Reagents. Phenylmethylsulfonyl fluoride (PMSF), benzamidine, leupeptin,pepstatin A, MgCl₂, EDTA, Trizma-Hydrochloride (Tris-HCl),(±)-propranolol and minimal essential medium (MEM) were obtained fromSigma Aldrich (St. Louis, Mo.). Egg phosphatidylcholine lipids (PC) wereobtained from Avanti Polar Lipids (Alabaster, Ala.). (±)-fenoterol waspurchased from Sigma Aldrich and [³H]-(±)-fenoterol was acquired fromAmersham Biosciences (Boston, Mass.). The organic solvents n-hexane,2-propanol and triethylamine were obtained as ultra pure HPLC gradesolvents from Carlo Erba (Milan, Italy). Fetal bovine serum andpenicillin-streptomycin were purchased from Life Technologies(Gaithersburg, Md.), and [¹²⁵I]-(±)-iodocyanopindolol (ICYP) waspurchased from NEN Life Science Products, Inc. (Boston, Mass.).

Preparation and Identification of (R,R)-fenoterol and (S,S)-fenoterol

(R,R)-fenoterol and (S,S)-fenoterol were prepared from (±)-fenoterolusing chiral HPLC techniques employing an HPLC column (25 cm×0.46 cmi.d.) containing the amylose tris-(3,5-dimethylphenylcarbamate) chiralstationary phase (CHIRALPAK® AD CSP, Chiral Technologies, West Chester,Pa.; CHIRALPAK is a registered trademark of Daicel Chemical IndustriesLtd., Exton, Pa.). The chromatographic system consisted of a JASCO®PU-980 solvent delivery system, and a JASCO® MD-910 multi-wavelengthdetector set at λ=230 nm, connected to a computer workstation; JASCO isa registered trademark of JASCO, Inc., Tokyo, Japan). A Rheodyne model7125 injector with 20 μl sample loop was used to inject 0.2-0.3 mg(±)-fenoterol onto the chromatographic system. The mobile phase wasn-hexane/2-propanol (88/12 v/v) with 0.1% triethylamine, the flow ratewas 1 mL/minute and the temperature of the system was maintained at 25°C. using a column heater/chiller (Model 7955, Jones Chromatography Ltd.,UK). The separated (R,R)-fenoterol and (S,S)-fenoterol were collected in10-mL fractions as the respective peaks eluted from the chromatographiccolumn. A 2-mL intermediate fraction was collected and discarded toimprove the enantiomeric purity of the collected isomers.

The stereochemical configurations of the resolved (R,R)-fenoterol and(S,S)-fenoterol were established using circular dichroism (CD)measurements obtained with a JASCO® J-800 spectropolarimeter. The(R,R)-fenoterol and (S,S)-fenoterol were dissolved in 2-propanol and themeasurements were obtained using 1 cm path length at room temperature.

Immobilized β2-AR Frontal Chromatography.

The liquid chromatography column containing the immobilized β2-AR wasprepared using a previously described technique (Beigi et al., Anal.Chem., 76: 7187-7193, 2004). In brief, cellular membranes were obtainedfrom a HEK 293 cell line that had been transfected with cDNA encodinghuman β2-AR. An aliquot of a cell pellet suspension corresponding to 5-7mg total protein, as determined by the micro BCA method, was used tocreate the column. The membranes were prepared in 10 mL buffer composedof Tris-HCl [50 mM, pH 7.4] containing MgCl₂ (2 mM), benzamidine (1 mM),leupeptin (0.03 mM) pepstatin A (0.005 mM) and EDTA (1 mM).

A 180 mg aliquot of immobilized artificial membrane chromatographicsupport (IAM-PC, 12 micron particle size, 300 Å pore size obtained fromRegis Chemical Co., Morton Grove, Ill.) and 80 μM PC were added to themembrane preparation and the resulting mixture was stirred at roomtemperature for 3 hours, transferred into (5 cm length) nitrocellulosedialysis membrane (MW cutoff 10,000 Da, Pierce Chemical, Rockford, Ill.)and placed in 1 L of dialysis buffer composed of Tris-HCl [50 mM, pH7.4] containing EDTA (1 mM), MgCl₂ (2 mM), NaCl (300 mM) and PMSF (0.2mM) at 4° C. for 24 hours. The dialysis step was repeated twice usingfresh buffer.

After dialysis, the mixture was centrifuged at 120×g for 3 minutes, thesupernatant was discarded and the pellet of IAM support containing theimmobilized receptor-bearing membranes was collected. The pellet wasresuspended in 2 mL chromatographic running buffer, composed of Tris-HCl[10 mM, pH 7.4] containing EDTA (1 mM) and MgCl₂ (2 mM) and thesuspension was pumped through a HR 5/2 chromatographic glass column(Amersham Pharmacia Biotech, Uppsala, Sweden) at a flow rate of 0.3mL/minutes using a peristaltic pump. The end adaptors were assembledproducing a total gel-bed volume of 0.4 mL. The column was stored at 4°C. when not in use.

The column containing the immobilized β2-AR stationary phase was placedin a chromatographic system composed of a HPLC pump (10-AD, ShimadzuInc., Columbia, Md.), a manually controlled FPLC injector (AmershamBiotechnology, Uppsala, Sweden) with a 50 μL sample loop, the packedimmobilized receptor column and an on-line radioactive flow detector(IN/US, Tampa, Fla.), all connected sequentially. In the frontalchromatographic studies, sample volumes of 5-7 mL were appliedcontinuously until the elution profile showed a plateau region. Therunning buffer was composed of Tris-HCl [10 mM, pH 7.4] containing EDTA(1 mM) and MgCl₂ (2 mM) and 0.05 nM [³H]-(±)-fenoterol, the markerligand. (R,R)-fenoterol or (S,S)-fenoterol was added to the runningbuffer in sequential concentrations of 0.1, 80.0, 240, and 700 nM, andapplied to the column. The immobilized receptor column was equilibratedwith about 80 mL of running buffer, without the added (R,R)-fenoterol or(S,S)-fenoterol respectively, in between each sample injection. Allchromatographic studies were carried out at room temperature at a flowrate of 0.2 mL/minutes.

The data were analyzed to determine the number of binding sites anddissociation constant using the non-linear equation (1),

$\begin{matrix}{{\lbrack M\rbrack \left( {{Vi}\text{-}V\mspace{14mu} \min} \right)} = \frac{P\mspace{14mu}\lbrack M\rbrack}{{Kd} + \lbrack M\rbrack}} & \left( {{Eq}.\mspace{14mu} 1} \right)\end{matrix}$

where V_(i) is the solute elution volume, V_(min) is the elution volumeat the saturation point, P is the number of available binding sites, Mis the concentration of the marker ligand and K_(d) is the dissociationconstant of the ligand.

Ligand-Displacement Binding.

Twenty-four hours after adenoviral infection with human β2-AR, HEK293cells were harvested in lysis buffer, Tris-HCl [5 mM, pH 7.4] containingEGTA [5 mM], and homogenized with 15 strokes on ice. Samples werecentrifuged at 30,000×g for 15 minutes to pellet membranes. Membraneswere resuspended in binding buffer, Tris-HCl [20 mM, pH 7.4] containingNaCl (120 mM), KCl (5.4 mM), CaCl₂ (1.8 mM), MgCl₂ (0.8 mM), and glucose(5 mM) and stored in aliquots at −80° C. Binding assays were performedon 5-10 μg of membrane protein using saturating amounts (1-300 pM) ofthe β-AR-specific ligand [¹²⁵I]cyanopindolol (ICYP). For competitionbinding, the 5-10 μg of membrane protein were pretreated with 50 μM ofGTP_(γs) (non-hydrolyzable guanosine triphosphate) and then incubatedwith ¹²⁵ICYP (50 pM) and different concentrations of fenoterol or itsisomers in a total volume of 250 Nonspecific binding was determined inthe presence of 20 μM propranolol. Reactions were conducted in 250 μL ofbinding buffer at 37° C. for 1 hour. The binding reaction was terminatedby addition of ice-cold Tris-HCl [10 mM, pH 7.4] to the membranesuspension, followed by rapid vacuum filtration through glass-fiberfilters (Whatman GF/C). Each filter was washed three times with anadditional 7 mL of ice-cold Tris-HCl [10 mM, pH 7.4]. The radioactivityof the wet filters was determined in a gamma counter. All assays wereperformed in duplicate, and receptor density was normalized tomilligrams of membrane protein. K_(d) and the maximal number of bindingsites (B_(max)) for ICYP were determined by Scatchard analysis ofsaturation binding isotherms. Data from competition studies wereanalyzed using 1- or 2-site competition binding curves with GRAPHPADPRISM® Software (GRAPHPAD PRISM is a registered trademark of GraphPadSoftware, Inc., San Diego, Calif.).

Example 2 Purification and Identification of (R,R)-fenoterol and(S,S)-fenoterol

This example demonstrates the resolution of (R,R)-fenoterol and(S,S)-fenoterol from (±)-fenoterol to a high degree of enantiomericpurity.

Using the chromatographic conditions described in Example 1,(±)-fenoterol was separated into its component enantiomers,(R,R)-fenoterol and (S,S)-fenoterol, on the AD-CSP. As illustrated inFIG. 1, the two stereoisomers were resolved with enantioselectivityfactor (a) of 1.21 and a resolution factor (R_(S)) of 1.06. Because ofthe observed tailing of the chromatographic peaks, a 2-mL intermediatefraction was collected and discarded. The collected peaks were analyzedusing the same chromatographic conditions and the data demonstrated thatboth enantiomers had been prepared with >97% stereochemical purity.

The assignment of the absolute configuration of the isolated fractionswas accomplished using their chiroptical properties. The ultraviolet(UV) spectra of both fractions contained identical maxima at about 280and 230, indicating the same UV chromophores for the two enantiomers.The circular dichroism (CD) spectrum shows, for the less retainedenantiomeric fraction, negative CD bands at about 280 and 215 nm, whilethe spectrum is positive at 230 and 200 nm. The sign of the CD bands isreversed for the most retained fenoterol fraction, this confirming theenantiomeric nature of the two fractions. The lowest energy UV and CDspectra of the two enantiomeric fractions are presented in FIGS. 2A and2B, respectively. The less retained chromatographic fraction showed anegative CD band at about 280 nm, while the CD spectrum of the mostretained chromatographic fraction contained a positive CD band at thesame wavelength (FIG. 2B). These results indicate that each of thefractions contained one of the fenoterol enantiomers.

The sign of the lowest energy CD band can be used to assign the absoluteconfiguration to the separated fenoterol enantiomers, by applying theBrewster-Buta/Smith-Fontana sector rule for chiral benzylic derivatives(Brewster and Buta, J. Am. Chem. Soc., 88: 2233-2240, 1996). This sectorrule is used to predict the sign of the CD band related to the ¹L_(b)electronic transition of the benzylic compounds, with either hydroxyl oramine moieties and has been primarily applied to conformationally mobilearomatic compounds containing a single stereogenic center. In the caseof fenoterol, there are two stereogenic centers. However, it is believedthat the observed optical activity is mainly determined by thearylcarbinol moiety, because of the distance between the aromatic ringand the stereogenic center. The application of this rule permitted theassignment of the absolute configuration of (S,S) to the fenoterolenantiomer contained in the less retained fraction that showed anegative CD band at 280 nm, and the absolute configuration of (R,R) tothe fenoterol enantiomer contained in the most retained fraction thatshowed a positive CD band at 280 nm. This assignment was confirmed bythe independent synthesis of (S,S)-fenoterol and (R,R)-fenoterol.

These studies indicate that (R,R)-fenoterol and (S,S)-fenoterol can beseparated from (±)-fenoterol to a high degree of enantiomeric purity.

Example 3 Chromatographic Determination of the Binding of(R,R)-fenoterol and (S,S)-fenoterol to the Immobilized β2-AR

This example demonstrates that (R,R)-fenoterol is responsible for theβ2-AR binding of the clinically used drug (±)-fenoterol.

The preparation, characterization and application of a liquidchromatographic stationary phase containing immobilized membranesobtained from the β2-AR HEK-293 cell line have been previously reported(Beigi et al., Anal. Chem., 76: 7187-7193, 2004). For example, Beigi etal. (Anal. Chem., 76: 7187-7193, 2004) demonstrated that frontaldisplacement chromatography could be used to determine the dissociationconstants (K_(d)) for the binding of two β2-AR antagonists,(S)-propranolol and CGP 12177A, to the immobilized β2-AR. Zonaldisplacement chromatography using CGP 12177A as the marker liganddemonstrated that the immobilized β2-AR had retained itsenantioselectivity as the addition of (S)-propranolol to the mobilephase produced a greater displacement than the addition of(R)-propranolol (Id.). The addition of (±)-fenoterol to the mobile phasewas also shown to produce a conformation change in the immobilized β2-AR(Id.). Agonist-induced conformational changes of the β2-AR, as well asmost G-protein coupled receptors, from a resting to active state havebeen documented (Ghanoui et al., Proc. Natl. Acad. Sci. U.S.A., 98:5997-6002, 2001).

Presently, the immobilized β2-AR column was equilibrated with therunning buffer containing the [³H]-fenoterol, the marker ligand, beforethe initiation of the displacement studies. It was assumed that thebinding data calculated using frontal displacement chromatographyreflects the binding of (R,R)-fenoterol and (S,S)-fenoterol to theactive state of the receptor. In frontal chromatography, the initialflat portion of the chromatographic trace represents the binding of amarker ligand that is specific for the immobilized target, in this studythe β2-AR, as well as non-specific binding to other sites on theimmobilized membrane fragments. Saturation of specific binding sitesproduces a breakthrough front followed by a plateau representing theestablishment of a new equilibrium. The addition of a second compoundinto the mobile phase will produce a shift of the chromatographic traceto the left if the compound competes with the marker for binding to theβ2-AR. The relationship between the magnitude of this shift and theconcentration of the marker ligand can be used to calculate the bindingaffinity of the displacer for the target and the number of activebinding sites. This approach has been recently reviewed (Moaddel andWainer, Anal. Chem. Acata, 546: 97-105, 2006).

As shown in FIG. 3 (curve 1), the addition of [³H]-fenoterol to therunning buffer produced the expected frontal chromatography trace.Sequential addition of increasing concentrations of (R,R)-fenoterol tothe running buffer produced a corresponding shift in the chromatographictraces towards smaller retention volumes (FIG. 3, curves 2-4). Themagnitude and the shift and the corresponding concentrations of(R,R)-fenoterol were analyzed using Eqn 1 and the calculateddissociation constant, K_(d), was 472 nM and the amount of availablebinding sites [P] was 176 pmoles per column, r²=0.9999 (n=2).

The sequential addition of increasing concentrations of (S,S)-fenoterolto the running buffer produced no corresponding shift in thechromatographic traces toward shorter retention times. Thus,(S,S)-fenoterol had no significant affinity for the immobilized β2-AR.

In order to validate the chromatographic results, a standard membranebinding study was conducted using membranes obtained from the sameHEK-293 cell line used to create the immobilized β2-AR column. The datareflected the presence of a single binding site with (mean±SD)K_(d)=457±55 nM (n=4) for (R,R)-fenoterol and 109,000±10,400 nM (n=4)for (S,S)-fenoterol. These data indicate that frontal affinitychromatography on immobilized cellular membrane columns can be used todetermine the magnitude and enantioselectivity of ligand binding to thetarget receptor. Further, the results from the frontal affinitychromatography and ligand competition binding studies both demonstratethat (R,R)-fenoterol is responsible for the β2-AR binding of theclinically used drug (±)-fenoterol.

Example 4 The effect of (R,R)-fenoterol and (S,S)-fenoterol onCardiomyocyte Contractility

This example demonstrates that (R,R)-fenoterol and (S,S)-fenoteroldifferentially activate β2-adrenergic receptor/stimulatoryheterotrimeric G protein (AR/G_(s)) signaling in regards to cellcontractility.

To determine if (R,R)-fenoterol and (S,S)-fenoterol differentiallyactivate β2-AR/G_(s) signaling in the regulation of cell contractility,freshly isolated adult rat cardiomyocytes were perfused with variousconcentrations of either (R,R)-fenoterol or (S,S)-fenoterol. Thesestudies were carried out using a previously described approach (Zhou etal., Mol. Pharmacol., 200, 58: 887-894). In brief, single ventricularmyocytes were isolated from 2-4 month old rat hearts by a standardenzymatic technique. The isolated cells were resuspended in HEPES buffersolution [20 mM, pH 7.4] containing, NaCl (137 mM), KCl (5.4 mM), MgCl₂(1.2 mM), NaH₂PO₄ (1.0 mM), CaCl₂ (1.0 mM), and glucose (20 mM). Allstudies were performed within 8 hours of cell isolation.

The cells were placed on the stage of an inverted microscope (Zeissmodel IM-35, Zeiss, Thornwood, N.Y.), perfused with the HEPES-bufferedsolution at a flow rate of 1.8 mL/minutes, and electrically stimulatedat 0.5 Hz at 23° C. Cell length was monitored by an opticaledge-tracking method using a photodiode array (Model 1024 SAQ, Reticon,Boston, Mass.) with a 3 ms time resolution. Cell contraction wasmeasured by the percent shortening of cell length following electricalstimulation.

The addition of (R,R)-fenoterol (10⁻⁸ to 10⁻⁵ M) produced a markedlyelevated positive inotropic effect and a significant upward shift of thedose-response curve compared to (±)-fenoterol (FIG. 4A). This wasdemonstrated by an increase in the maximum contractile response from265±11.6% to 306±11.8% resting cell length (p<0.05) and a reduction inEC₅₀ from −7.0±0.2 to −7.1±0.2 log [M](p<0.05). In contrast,(S,S)-fenoterol had only a minor positive inotropic effect (FIG. 4B).

The cardiomyocyte contractility studies indicate that (R,R)-fenoterol isresponsible for the observed β2-AR agonist activity in cardiomyocytes.

Example 5 Synthesis

General Procedures:

All reactions were carried out using commercial grade reagents andsolvents. Tetrahydrofuran (THF) was dried by refluxing over sodium andbenzophenone. Dichloromethane was dried by refluxing over calciumhydride. Ultraviolet spectra were recorded on a Cary 50 Concentrationspectrophotometer. Optical rotations were done on a Rudolph ResearchAutopol IV. NMR Spectra were recorded on a Varian Mercury VMX 300-MHzspectrophotometer using tetramethylsilane as the internal standard. NMRmultiplicities were reported by using the following abbreviations: s,singlet; d, doublet; t, triplet; q, quartet; p, pentet; m, multiplet;apt., apparent; and br, broad. Low resolution mass spectra were obtainedon a Finnigan LCQ^(Duo) LC MS/MS atmospheric pressure chemicalionization (API) quadrupole ion trap MS system equipped with bothelectrospray (ESI) and atmospheric pressure chemical ionization (APCI)probes. Analytical HPLC data was obtained using a Waters 2690Separations Module with PDA detection. Method (a): ThermoHypersil BDS100×4.6 mm C18 column, H₂O/CH₃CN/TFA. Method (b): Brownlee PhenylSpheri-5 100×4.6 mm, water/acetonitrile/TFA. Method (c): Vydac 150×4 mmC18 column, H₂O/isopropanol/TFA. Method (d): CHIRALPAK® AD-H 250×10 mm,95/5/0.05 CH₃CN/isopropanol/diethylamine. Merck silica gel (230-400mesh) was used for open column chromatography.

3′,5′-Dibenzyloxy-α-bromoacetophenone (46)

A solution of 2.4 mL (46 mmol) of Br₂ in 45 mL of CHCl₃ was addeddropwise over 1 h to a chilled, stirring solution of 9.66 g (29 mmol) of3′,5′-dibenzyloxyacetophenone (45) in 40 mL of CHCl₃. The resultingsolution was allowed to warm to room temperature over 1 hour with goodstirring, then poured into 100 mL of cold H₂O and transferred to aseparatory funnel where the CHCl₃ fraction was isolated, washed withbrine solution, dried (Na₂SO₄), filtered, and concentrated to 10.8 g.This material was applied to 500 g of silica gel, eluting with CHCl₃ toobtain 2.65 g (22%) of compound 46 as a white solid. ¹H NMR (CDCl₃) δ4.39 (s, 2H), 5.08 (s, 4H), 6.85 (t, 1H, J=2.1 Hz), 7.20 (d, 2H, J=2.4Hz), 7.31-7.44 (m, 10H).

General procedure for the Enantioselective Reduction of compound 46 to3′,5′-Dibenzyloxyphenylbromohydrins [(R)-8(S)-8]

Under argon atmosphere, ˜0.06 mL (0.316 mmol, 10 mol %) of 5.0 Mboron-methyl sulfide complex (BH₃SCH₃) in diethyl ether was added in oneportion to a solution of 25 mg (0.16 mmol, 5 mol %) of the appropriatecis-1-amino-2-indanol in 3 mL of dry THF. This material under argon wasadded over 30 minutes to a solution of 1.3 g (3.16 mmol) of3′,5′-dibenzyloxy-α-bromoacetophenone in 20 mL of dry THF, while at thesame time adding in ˜0.05 mL pulses, 0.45 mL of 5.0 M boron-methylsulfide complex. The resulting solution was stirred under argon for 2hours, and then quenched with 3 mL of methanol, controlling gasevolution. Solvents were removed in vacuo and the resulting residuetaken up in 30 mL of CHCl₃ and washed with 25 mL of 0.2 M sulfuric acidfollowed by 20 mL of brine, then dried (Na₂SO₄), filtered, andevaporated.

(R)-(+3′,5′-Dibenzyloxyphenylbromohydrin [(R)-8]

Prepared with (1R,2S)-(+)-cis-1-amino-2-indanol as the enantioselectivereduction catalyst to give 1.02 g (78%) of (R)-8 as a fine white powder.¹H NMR (CDCl₃) δ 3.44 (dd, 1H, J=9.0, 10.5 Hz), 3.55 (dd, 1H, J=3.3,10.5 Hz), 4.79 (dd, 1H, J=3.3, 8.7 Hz), 4.97 (s, 4H), 6.51 (t, 1H, J=2.4Hz), 6.57 (d, 2H, J=1.8 Hz), 7.21-7.38 (m, 10H); [α]_(D)=−12.1° (c=1.0MeOH).

(S)-(+)-3′,5′-Dibenzyloxyphenylbromohydrin [(S)-8]

Prepared with (1S,2R)-(−)-cis-1-amino-2-indanol as the enantioselectivereduction catalyst to give 1.07 g (82%) of (S)-8 as a fine white powder.¹H NMR (CDCl₃) δ 3.43 (dd, 1H, J=9.0, 10.5 Hz), 3.55 (dd, 1H, J=3.3,10.5 Hz), 4.78 (dd, 1H, J=3.3, 8.7 Hz), 4.96 (s, 4H), 6.50 (t, 1H, J=2.4Hz), 6.57 (d, 2H, J=1.8 Hz), 7.21-7.39 (m, 1011); [α]_(D)=+11.8° (c=0.90MeOH).

4-Benzyloxyphenylacetone (34)

To 10.0 g (41.3 mmol) of 4-benzyloxyphenylacetic acid (31) was added, 20mL of acetic anhydride and 20 mL of pyridine, which was heated to refluxwith stirring under argon atmosphere for 6 hours. Solvents wereevaporated and residue dissolved in CHCl₃ (50 mL) and washed with 1NNaOH (2×50 mL). Dried organic layer (MgSO₄), filtered, and evaporated to11.8 g of an amber oil. Vacuum distillation at 0.1 mm Hg in an oil-bathset to 170° C. followed by silica gel chromatography eluting with 8/2CH₂Cl₂-hexanes gave 2.68 g (27%). ¹H NMR (CDCl₃) □δ□ 2.14 (s, 3H), 3.63(s, 2H), 5.05 (s, 2H), 6.94 (d, 21-1, J=8.7 Hz), 7.10 (d, 2H, J=8.7 Hz),7.26-7.47 (m, 5H).

Phenylacetone (35)

A solution of 20.4 g (0.15 mol) of phenylacetic acid, acetic anhydride(70 mL) and pyridine (70 mL) was heated to reflux with stirring underargon atmosphere for 6 hours. Solvents were evaporated and residuedissolved in CHCl₃ (100 mL), washed with 1N NaOH (2×100 mL) and driedthe organic layer (MgSO₄), filtered, and evaporated to give 20.4 g.Vacuum distillation at 0.1 mm Hg in an oil bath set to 160° C., followedby silica gel chromatography eluting with 1/1 hexanes/CH₂Cl₂ gave 5.5 g(27%). ¹H (CDCl₃) δ□ 2.15 (s, 3H), 3.70 (s, 2H), 7.20-7.36 (m, 5H).

1-Naphthalen-1-yl-propan-2-one (36)

A solution of 37.2 g (20 mmol) of naphthoic acid (33), acetic anhydride(100 mL) and pyridine (100 mL) was heated to reflux with stirring underargon atmosphere for 6 hours. Evaporated solvents, dissolved residue inCHCl₃ (200 mL) and washed with 1N NaOH (2×150 mL), dried organic layer(MgSO₄), filtered, and evaporated to give 34.6 g. Distillation at 0.5 mmHg in an oil bath set to 170° C., followed silica gel chromatographyeluting with 1/1 hexanes/CH₂Cl₂ gave 9.7 g (26%). ¹H (CDCl₃) δ□ 2.11 (s,3H), 4.12 (s, 2H), 7.40-7.53 (m, 4H), 7.81 (d, 1H, J=8.4 Hz), 7.87-7.90(m, 2H).

General Procedure for Preparation of 2-benzylaminopropanes (37-39, 42,43)

To the appropriate ketone (1 eq) in CH₂Cl₂ (c=0.5 M), cooled to 0° C.was added glacial HOAc (1 eq), followed by benzylamine (1 eq) andNaBH(AcO)₃ (1.4 eq). The reaction mixture was warmed to room temperatureand stirred under argon for 20 hours. The reaction mixture was cooled(ice bath), 10% NaOH (5 eq) was added dropwise and then extracted intoCH₂Cl₂, washed with brine. The product was then dried (Na₂SO₄), filteredand evaporated.

1-(4-benzyloxy)-2-benzylaminopropane (37)

Prepared from 4-benzyloxy-phenylacetone (34; 2.0 g, 8.3 mmol) to afford2.61 g (95%) as a tan solid. ¹H (CDCl₃) δ 1.10 (d, 3H, J=6.3 Hz),2.50-2.58 (m, 1H). 2.68-2.77 (m, 1H), 2.82-2.89 (m, 1H), 3.75 (dd, 2H,J=12 Hz, J=30 Hz), 5.05 (s, 2H), 6.90 (d, 2H, 0.1=8.7 Hz), 7.04 (d, 2H,J=8.7 Hz), 7.17-7.42 (m, 10H); MS (APCI+) m/z (rel): 332 (100).

1-Phenyl-2-benzylaminopropane (38)

Prepared from phenylacetone (35; 5.5 g, 41 mmol) to afford 8.4 g (91%)as a tan solid. ¹H (CDCl₃) δ□ 1.09 (d, 3H, J=6.3 Hz), 2.61-2.81 (m, 2H),2.92 (m, 1H), 3.80 (dd, 2H), 7.14-7.30 (m, 10H); MS (APCI+) m/z (rel):226 (100).

1-(1′-Naphthyl)-2-benzylaminopropane (39)

Prepared from 1-naphthalen-1-yl-propan-2-one (36; 5.0 g, 27.1 mmol) toafford 7.0 g (94%) as a tan solid. ¹H (CDCl₃) δ□ 1.14 (d, 3H, J=6.0 Hz),3.02-3.18 (m, 2H), 3.27 (m, 1H), 3.80 (dd, 2H, J=13.2, 43.8 Hz),7.13-7.23 (m, 5H), 7.31-7.48 (m, 4H), 7.73 (d, 1H, J=7.8 Hz), 7.83-7.86(m, 1H), 7.96-7.99 (m, 1H); MS (APCI+) m/z (rel): 276 (100).

1-(4′-Methoxyphenyl)-2-benzylaminopropane (42)

Prepared from 4-methoxyphenyl-acetone (40; 2.75 g, 131 mmol) to afford2.31 g (97%). ¹H (CDCl₃) δ□ 1.10 (d, 3H, J=6.3 Hz), 2.56-2.75 (m, 2H),2.90 (m, 1H), 3.79 (s, 1H), 3.79 (m, 2H, J=13.2 Hz), 6.82 (d, 2H, J=8.7Hz), 7.07 (d, 2H, J=8.7 Hz), 7.18-7.32 (m, 5H); MS (APCI+) m/z (rel):256 (100).

1-(4′-nitrophenyl)-2-benzylaminopropane (43)

Prepared from 4-nitrophenyl-acetone (41; 4.95 g, 28 mmol) to afford 7.32g (98%) as an amber oil. ¹H (CDCl₃) δ□ 1.60 (d, 3H, J=6.3 Hz), 2.73-2.85(m, 1H), 3.00-3.12 (m, 2H), 3.86 (dd, 2H, J=26 Hz, J=60 Hz), 7.23-7.40(m, 5H), 7.30 (d, 2H, J=9.0 Hz), 8.14 (d, 2H, J=8.7 Hz). MS (APCI+) m/z(rel): 271 (100).

General procedure for Enantiomeric Separation of 2-benzylaminopropanes[(R)-10-14(S)-10-14]

The appropriate racemic 2-benzylaminopropane (1 eq) was combined withthe appropriate optically active mandelic acid (1 eq) in methanol (c=0.5M) and refluxed until the solution homogenized, then cooled to RT. Thecrystals were filtered, collected, and recrystallized twice frommethanol (c=0.3 M) to afford the optically active2-benzylaminopropane.mandelic acid salt. The salts were converted to thefree amine for the purpose of collecting NMR and rotation data bypartitioning the mandelic acid salt between 10% K₂CO₃ and CHCl₃, dryingorganic extracts (Na₂SO₄) and evaporating.

(R)-(−)-1-(4′-benzyloxy)-2-benzylaminopropane [(R)-10]

A sample of 2.13 g (6.42 mmol) of 1-(4-benzyloxy)-2-benzylaminopropane(37) was reacted with 972 mg (6.42 mmol) of (R)-(−)-mandelic acid togive 295 mg (28% based on enantiomeric abundance) of the free amineafter workup. ¹H NMR (CDCl₃) δ□ 1.12 (d, 3H, J=6.3 Hz), 2.58-2.78 (m,2H), 2.82-2.91 (m, 1H), 3.75 (dd, 2H, J=12 Hz, J=30 Hz)), 5.07 (s, 2H),6.93 (d, 2H, J=8.7 Hz), 7.10 (d, 2H, J=8.7 Hz), 7.21-7.42 (m, 10H); MS(APCI+) m/z (rel): 332 (100); [α]_(D)=−19.1° (c=1.4, MeOH).

(S)-(+)-1-(4′-benzyloxy)-2-benzylaminopropane [(S)-10]

The washes from the separation of (R)-10 were concentrated andpartitioned between 50 mL of chloroform and 50 mL of 10% K₂CO₃ in water.Washed organics with brine, dried (Na₂SO₄), filtered and evaporated to1.70 g (5.1 mmol). The organics were brought to reflux with 782 mg (5.1mmol) of (S)-(+)-mandelic acid (as previously described) andcrystallized 3 times to obtain 670 mg of the (S)-amine.(S)-mandelic acidsalt. The (S)-amine.(S)-mandelic acid salt was triturated in ether thenpartitioned between 30 mL of chloroform and 20 mL of 10% K₂CO₃ in water.The organic partition was washed with brine, then dried (Na₂SO₄),filtered and evaporated to give 366 mg of the free amine (33% based onenantiomeric abundance). ¹H NMR (CDCl₃) δ□ 1.10 (d, 3H, J=6.3 Hz),2.58-2.78 (m, 2H), 2.82-2.91 (m, 1H), 3.76 (dd, 2H, J=12, 30 Hz), 5.06(s, 2H), 6.93 (d, 2H, J=8.7 Hz), 7.09 (d, 2H, J=8.7 Hz), 7.21-7.42 (m,10H); MS (APCI+) m/z (rel): 332 (100); [α]_(D)=+19.2° (c=1.5 MeOH).

(R)-(−)-1-(4′-Methoxyphenyl)-2-benzylaminopropane [(R)-11]

A sample of 3.02 g (11.8 mmol) of1-(4′-methoxyphenyl)-2-benzylaminopropane (42) was reacted with 1.8 g(11.8 mmol) (S)-(+)-mandelic acid to give 530 mg (35% based onenantiomeric abundance) of the free amine after workup. ¹H NMR (CDCl₃)δ□ 1.10 (d, 3H, J=6.3 Hz), 2.57-2.76 (m, 2H), 2.88-2.94 (m, 1H), 3.79(s, 3H), 3.72-3.88 (m, 2H), 6.82 (d, 2H, J=8.7 Hz), 7.07 (d, 2H, J=8.4Hz), 7.15-7.31 (m, 5H); MS (APCI+) m/z (rel): 256 (100); [α]_(D)=−30.4°(c=1.25 MeOH).

(S)-(+)-1-(4′-Methoxyphenyl)-2-benzylaminopropane [(S)-11]

A sample of 3.36 g (13.2 mmol) of the racemate1-(4′-methoxyphenyl)-2-benzylaminopropane (42) was reacted with 2.0 g(13.2 mmol) of (R)-(−)-mandelic acid to give 740 mg (44% based onenantiomeric abundance) of the free amine after workup. ¹H NMR, (CDCl₃)δ□ 1.10 (d, 3H, J=6.2 Hz), 2.55-2.76 (m, 2H), 2.88-2.95 (m, 1H),3.73-3.88 (m, 2H), 3.79 (s, 3H), 6.80 (d, 2H, J=8.7 Hz), 7.08 (d, 2H,J=8.4 Hz), 7.15-7.30 (m, 5H); MS (APCI+) m/z (rel): 256 (100);[α]_(D)=+30.5° (c=1.1 MeOH).

(R)-(−)-1-(4′-nitrophenyl)-2-benzylaminopropane [(R)-12]

A sample of 2.0 g (7.3 mmol) of 1-(4′-nitrophenyl)-2-benzylaminopropane(43) was reacted with 1.13 g (7.3 mmol) of (S)-(+)-mandelic acid to give486 mg (49% based on enantiomeric abundance) of the free amine afterworkup. ¹H NMR (CDCl₃) δ□ 1.60 (d, 3H, J=6.3 Hz), 2.73-2.85 (m, 1H),3.00-3.12 (m, 2H), 3.86 (dd, 2H, J=26 Hz, J=60 Hz), 7.23-7.40 (m, 5H),7.30 (d, 2H, J=9.0 Hz), 8.14 (d, 2H, J=8.7 Hz); MS (APCI+) m/z (rel):271 (100); [α]_(D)=−9.3° (c=1.0 MeOH).

(S)-(+)-1-(4′-nitrophenyl)-2-benzylaminopropane [(S)-12]

A sample of 2.0 g (7.3 mmol) of 1-(4′-nitrophenyl)-2-benzylaminopropane(43) was reacted with 1.13 g (7.3 mmol) of (R)-(−)-mandelic acid to give640 mg (65% based on enantiomeric abundance) of the free amine afterworkup. ¹H NMR (CDCl₃) δ□ 1.60 (d, 3H, J=6.3 Hz), 2.73-2.85 (m, 1H),3.00-3.12 (m, 2H), 3.86 (dd, 2H, J=26, 60 Hz), 7.23-7.40 (m, 5H), 7.30(d, 2H, J=9.0 Hz), 8.14 (d, 2H, J=8.7 Hz); MS (APCI+) m/z (rel): 271(100); [α]_(D)=+8.2° (c=1.0 MeOH).

(R)-(−)-1-Phenyl-2-benzylaminopropane [(R)-13]

A sample of 2.62 g (11.6 mmol) of 1-phenyl-2-benzylaminopropane (38) wasreacted with 1.77 g (11.6 mmol) of (S)-(+)-mandelic acid to give 747 mg(57% based on enantiomeric abundance) of the free amine after workup. ¹HNMR (CDCl₃) δ□□ 1.13 (d, 3H, J=6.0 Hz), 2.62-2.84 (m, 2H), 2.92-2.99 (m,1H), 3.81 (dd, 2H, J=13.2, 34.5 Hz) 7.14-7.29 (m, 10H); MS (APCI+) m/z(rel): 226 (100); [α]_(D)=−24.5° (c=1.10 MeOH).

(S)-(+)-1-Phenyl-2-benzylaminopropane [(S)-13]

A sample of 5.0 g (22.2 mmol) of racemic 1-phenyl-2-benzylaminopropane(38) was reacted with 3.4 g (22.2 mmol) of (R)-(−)-mandelic acid to give2.15 g (86% based on enantiomeric abundance) of the free amine afterworkup. ¹H NMR (CDCl₃) δ 1.11 (d, 3H, J=6.0 Hz), 2.62-2.84 (m, 2H),2.92-2.99 (m, 1H), 3.81 (dd, 2H, J=13.2, 34.5 Hz), 7.14-7.29 (m, 5H); MS(APCI+) m/z (rel): 226 (100); [α]_(D)=+18.2° (c=0.85 MeOH).

(R)-(−)-1-(1′-Naphthyl)-2-benzylaminopropane [(R)-14]

The washes recovered from the separation of (S)-14 were concentrated andpartitioned between 40 mL of chloroform and 40 mL of 10% K₂CO₃ in water.The organic partition was washed with 20 mL of brine then dried (Na₂SO₄)to afford 1.16 g (4.2 mmol) of the free amine, which was reacted with640 mg (4.2 mmol) of (S)-(+)-mandelic acid. 588 mg (46% based onenantiomeric abundance) of the free amine was obtained. ¹H NMR (CDCl₃)δ□ 1.07 (d, 3H, J=6.0 Hz), 3.02-3.18 (m, 2H), 127 (m, 1H), 3.74 (dd, 2H,J=13.2, 30.9 Hz), 7.13-7.23 (m, 5H), 7.31-7.48 (m, 4H), 7.73 (d, 1H,J=7.8 Hz), 7.83-7.86 (m, 1H), 7.96-7.99 (m, 1H); MS (APCI+) m/z (rel):276 (100); [α]_(D)=−5.8° (c=1.0 MeOH).

(S)-(+)-1-(1′-Naphthyl)-2-benzylaminopropane [(S)-14]

A sample of 2.6 g (9.4 mmol) of 1-(1′-naphthyl)-2-benzylaminopropane(39) was reacted with 1.44 g (9.4 mmol) of (R)-(−)-mandelic acid to give420 mg (21% based on enantiomeric abundance) of the free amine afterworkup. ¹H NMR (CDCl₃) δ□ 1.07 (d, 3H, J=6.0 Hz), 3.02-3.18 (m, 2H),3.27 (m, 1H), 3.74 (dd, 2H, J=13.2, 30.9 Hz), 7.13-7.23 (m, 5H),7.31-7.48 (m, 4H), 7.73 (d, 1H, J=7.8 Hz), 7.83-7.86 (m, 1H), 7.96-7.99(m, 1H); MS (APCI+) m/z (rel): 276 (100); [α]_(D)=+6.3° (c=1.0 MeOH).

(R)-(−)-2-Benzylaminoheptane [(R)-15]

A sample of 0.65 mL (4.4 mmol) of (R)-(−)-2-aminoheptane (R-44) 0.44 mL(4.4 mmol) of benzaldehyde and 0.1 mL of HOAc were combined in 40 mL ofCH₂Cl₂ and then cooled to 0° C. To the reaction mixture was added 2.75mg (13 mmol) of sodium triacetoxyborohydride in one portion, which wasstirred under argon at room temperature for 28 hours. The reactionmixture was diluted with 30 mL of CH₂Cl₂, cooled in an ice bath and 80mL of 5% NaOH (in water) was added. Fractions were separated, organics(Na₂SO₄) dried and evaporated to 638 mg (71%) of (R)-15. ¹H NMR (CDCl₃)δ 0.88 (m, 3H), 1.08 (d, 3H J=6.6 Hz), 1.20-1.39 (m, 6H), 1.41-1.67 (m,2H), 3.62-3.77 (m, 1H), 3.75 (p, 2H, J=12 Hz), 7.17-7.41 (m, 5H); MS(APCI+) m/z (rel): 206 (100); [α]_(D)=+6.9° (c=1.0, MeOH).

(S)-(+)-2-Benzylaminoheptane [(S)-15]

A sample of 0.15 mL (1 mmol) of (S)-(+)-2-aminoheptane ((S)-44) 0.1 mL(1 mmol) of benzaldehyde and 0.1 mL of HOAc were combined in 10 mL ofCH₂Cl₂ and cooled to 0° C., then added 650 mg (3 mmol) of sodiumtriacetoxyborohydride in one portion. The reaction mixture was stirredunder argon at room temperature for 28 hours. The mixture was dilutedwith 10 mL of dichloromethane, cooled in an ice bath and 20 mL of 5%NaOH (in water) was added. Fractions were separated, organics were dried(Na₂SO₄) and evaporated to 154 mg (70%). ¹H NMR (CDCl₃) δ□ 0.88 (m, 3H),1.08 (d, 3H J=6.6 Hz), 1.19-1.37 (m, 6H), 1.41-1.67 (m, 2H), 3.62-3.77(m, 1H), 3.75 (p, 2H, J=12 Hz), 7.17-7.41 (m, 5H); MS (APCI+) m/z (rel):206 (100); [α]_(D)=+7.8° (c=1.0, MeOH).

Preparation of Fenoterol Analogs, Procedure A

To form the epoxide, the appropriate 3′,5′-dibenzyloxyphenylbromohydrin((R)-8) or (S)-8 (1 eq) was combined with K₂CO₃ (1.4 eq) in 1:1 THF/MeOH(c=0.3 M) and stirred for 2 hours under argon at room temperature. Thesolvent was removed and the residue partitioned between toluene and H₂O.The toluene fraction was isolated, dried (Na₂SO₄), filtered, andevaporated. The residue was dissolved with the appropriate freebenzylamine (R)- or (S)-10-15, 28 (0.95 eq) in a good amount of tolueneand evaporated again under high vacuum to remove trace H₂O. Theresulting colorless residue was heated to 120° C. under argon for 20hours, cooled and checked by ¹H NMR and mass spectrometry to confirmcoupling. The residue was dissolved in EtOH (c=0.07 M) with heat andtransferred to a Parr flask, where it was hydrogenated at 50 psi ofhydrogen over 10% (wt) Pd/C (10 mg cat/65 mg bromohydrin) for 24 hours.Complete debenzylation was confirmed by mass spectrometry. The mixturewas filtered through Celite, the filter cake rinsed with isopropanol,and the filtrate concentrated. The residue was dissolved in 1:1isopropanol/EtOH (c=0.2 M) and brought to reflux for 30 minutes with 0.5eq of fumaric acid. The reaction was cooled and the solvent removed. Thecrude material was purified by open column chromatograph or preparativechromatograph.

Column Separation of (R,R)-1 and (S,S)-1, Procedure B

A sample of 75 mg of fenoterol HBr was dissolved in 1.5 mL of 95/5/0.05CH₃CN/isopropanol/HNEt₂ and applied in 100 μL injections to a CHIRALPAK®AD-H 10×250 mm 5 μm semi-preparative column using a waters 2690Separations Module, PDA set to 280 nm. The eluting solvent was 95/5/0.05CH₃CN/isopropanol/HNEt₂, 5 mL/min. Retention times for (S,S) and (R,R)isomers were 4.8 min and 7.8 min, respectively.

(R,R)-(−)-Fenoterol [(R,R)-1]

Obtained according to Procedure B to give 40 mg collected afterevaporation. ¹H NMR (CD₃OD) δ 1.05 (d, 3H, J=6.3 Hz), 2.49 (q, 1H, J=6.9Hz), 2.62-2.74 (m, 2H), 2.80-2.91 (m, 2H), 4.55 (dd, 1H, J=5.1, J=3.3Hz), 6.16 (t, 1H, J=2.4 Hz), 6.27 (d, 2H, J=2.1 Hz), 6.68 (d, 2H, J=8.4Hz), 6.94 (d, 2H, J=8.4 Hz); ¹³C NMR (CD₃CN) δ 20.3, 43.2, 55.1, 55.2,72.4, 102.2, 105.4, 116.0, 131.3, 131.8, 147.4, 156.2, 159.0; UV (MeOH)λ_(max) 279 nm (∈ 2,760), 225 (12,900), 204 (32,600); MS (APCI+) m/z(rel): 304 (100, M+H); [α]_(D)=−29.0° (conc=0.2% MeOH); HPLC: (a) 0.1%diethylamine in H₂O, 0.50 mL/min, 254 nm, t_(R) 2.90 min, 99% pure; (d)t_(R) 7.8 min, >99% pure.

(S,S)-(+)-Fenoterol [(S,S)-1]

Obtained according to Procedure B to give 35 mg after evaporation. ¹HNMR (CD₃OD) δ 1.05 (d, 3H, J=6.6 Hz), 2.49 (q, 1H, J=7.2 Hz), 2.62-2.76(m, 2H), 2.80-2.94 (m, 2H), 4.55 (dd, 1H, J=4.8, J=3.3 Hz), 6.16 (t, 1H,J=2.1 Hz), 6.27 (d, 2H, J=2.4 Hz), 6.68 (d, 2H, J=8.4 Hz), 6.94 (d, 2H,J=8.4 Hz); ¹³C (CD₃CN) δ 20.3, 43.2, 55.0, 55.2, 72.4, 102.2, 105.4,116.0, 131.3, 131.8, 147.4, 156.2, 159.0; UV (MeOH) λ_(max) 279 nm(∈2,680), 224 (12,700), 204 (32,800); MS (APCI+) m/z (rel): 304 (100,M+H); [α]_(D)=+28.5° (conc=0.20% MeOH); HPLC: (a) 0.1% diethylamine inH₂O, 0.50 mL/min, 254 nm, t_(R) 2.72 min, >99% pure; (d) t_(R) 4.8min, >99% pure.

(R,S)-(−)-Fenoterol Fumarate [(R,S)-1]

Prepared from (R)-8 and (S)-10 according to Procedure A to give 168 mg(64%). ¹H NMR (CD₃OD) δ 1.22 (d, 3H, J=6.6 Hz), 2.64 (dd, 1H, J=9.9 Hz,J=13.2 Hz), 3.01-3.51 (m, 4H), 4.79 (dd, 1H, J=3.0 Hz, J=9.9 Hz), 6.23(t, 1H, J=2.4 Hz), 6.36 (d, 2H, J=2.1 Hz), 6.75 (s, 1H), 6.76 (d, 2H,J=8.4 Hz), 7.05 (d, 2H, J=8.1 Hz); ¹³C NMR (CD₃OD) δ 16.2, 39.1, 52.5,57.4, 70.4, 103.4, 105.3, 116.7, 127.8, 131.4, 135.2, 144.6, 157.7,160.0, 168.2; UV (MeOH) λ_(max) 278 nm (∈ 2,520), 205 (27,900); MS(ESI+) m/z (rel): 304 (100, M+H); [α]_(D)=−7.5° (conc=0.75% MeOH); HPLC:(a) 70/30/0.05. 1.00 mL/min, 282 nm, t_(R) 1.35 min, >99% pure; (b)50/50/0.05. 1.0 ml, 0.50 mL/min, 254 nm, t_(R) 2.72 min, >99% pure; (d)t_(R) 4.8 min, 1.00 mL/min, 280 nm, t_(R) 2.10 min, 97.5% pure.

(S,R)-(+)-Fenoterol Fumarate [(S,R)-1]

Prepared from (S)-8 and (R)-10 according to Procedure A to give 104 mg(39%). ¹H NMR (CD₃OD) δ 1.22 (d, 3H, J=6.6 Hz), 2.64 (dd, 1H, J=9.9 Hz,J=13.5 Hz), 3.47-3.04 (m, 4H), 4.80 (dd, 1H, J=2.7, J=9.6 Hz), 6.23 (t,1H, J=2.4 Hz), 6.36 (d, 2H, J=2.1 Hz), 6.75 (s, 1H), 6.76 (d, 2H, J=8.4Hz), 7.05 (d, 2H, J=8.4 Hz); ¹³C NMR (CD₃OD) δ 16.2, 39.1, 52.5, 57.4,70.4, 103.4, 105.3, 116.7, 127.8, 131.4, 135.2, 144.6, 157.7, 159.9,168.2; UV (MeOH) λ_(max) 278 nm (∈ 2,640), 202 (36,600); MS (ESI+) m/z(rel): 304 (100, M+H), 413 (10); [α]_(D)=+6.4° (conc=0.50% MeOH); HPLC:(a) 70/30/0.05, 1.00 mL/min, 282 nm, t_(R) 1.35 min, 95.9% pure; (b)50/50/0.05, 1.0 mL/min, 280 nm, t_(R) 2.06 min, 99% pure.

(R,R)-(−)-1-p-Methoxyphenyl-2-(β-3′,5′-dihydroxy-phenyl-β-oxy)ethylamino-propaneFumarate [(R,R)-2]

Prepared from (R)-8 and (R)-11 according to Procedure A to give 172 mg(38%). ¹H NMR (CD₃OD) δ 1.08 (d, 3H, J=6.3 Hz), 3.05-2.56 (m, 5H), 4.57(dd. 1H, J=8.4, 5.4 Hz), 6.16 (m, 1H), 6.26 (d, 2H, J=2.7 Hz), 6.81 (d,2H, J=8.7 Hz), 7.03 (d, 2H J=8.7 Hz); ¹³C NMR (CD₃OD) δ 18.8, 42.3,54.5, 55.6, 56.0, 72.6, 103.0, 105.4, 115.0, 131.1, 131.2, 131.3, 146.2,159.8, 159.9; UV (MeOH) λ_(max) □□277 nm (∈ 3,590), 224 (17,700), 207(29,500); MS (ESI+) m/z (rel): 318 (100, M+H); [α]_(D)=−24.9° (c=0.8MeOH); HPLC: (a) 70/30/0.05, 1.0 mL/min, 282 nm, t_(R) 1.54 min, 96.5%pure; (b) 50/50/0.05, 2.0 mL/min, 276 nm, t_(R) 1.51 min, 95.9% pure.

(S,S)-(+)-1-p-Methoxyphenyl-2-(β-3′,5′-dihydroxy-phenyl-β-oxy)ethylamino-propaneFumarate [(S,S)-2]

Prepared from (S)-8 and (8)-11 according to Procedure A to give 318 mg(53%). ¹H NMR (CD₃OD) δ 1.15 (d, 3H, J=6.0 Hz), 2.58-3.22 (m, 5H), 3.77(s, 3H), 4.68 (dd, 1H, J=4.8, 8.4 Hz), 6.18 (t, 1H, J=2.1 Hz), 6.31 (d,2H, J=2.1 Hz), 2.23 (s, 0.5H, fumarate), 6.84 (d, 2H, J=8.7 Hz), 7.10(d, 2H, J=9.0 Hz); ¹³C NMR (CD₃OD) δ 16.1, 39.9, 52.4, 54.5, 55.3, 70.4,101.9, 104.2, 114.0, 129.2, 130.1, 144.4, 158.7, 158.9; UV (MeOH)λ_(max) 277 nm (∈ 2,100), 224 (11,00), 205 (22,700); MS (ESI+) m/z(rel): 318 (100, M+H); [α]_(D)=+28.6° (c=0.95 MeOH); HPLC: (a)70/30/0.05, 1.0 mL/min, 282 nm, t_(R) 1.67 min, 96.0% pure; (b)50/50/0.05, 2.0 mL/min, 276 nm, t_(R) 1.51 min, 97.1% pure.

(R,S)-(+1-p-Methoxyphenyl-2-(β-3′,5′-dihydroxy-phenyl-β-oxy)ethylaminopropaneFumarate [(R,S)-2]

Prepared from (R)-8 and (S)-11 according to Procedure A to give 160 mg(38%). ¹H NMR (CD₃OD) δ 1.20 (d, 3H, J 6.6 Hz), 2.62-2.71 (m, 1H),2.98-3.20 (m, 3H), 3.30-3.42 (m, 2H), 4.73-4.81 (m, 1H), 6.21 (m, 2H),3.35 (m, 2H), 6.71 (s, 0.5H, fumarate), 6.56-6.89 (m, 2H), 7.11-7.19 (m,2H); ¹³C NMR (CD₃OD) δ 15.6, 38.5, 51.8, 54.5, 55.9, 69.7, 102.1, 104.1,114.1, 128.5, 130.2, 136.0, 143.8, 158.8, 159.1; UV (MeOH) λ_(max) 277nm (∈ 4,100), 224 (21,400), 203 (50,600); MS (ESI+) m/z (rel): 318 (100,M+H); [60]_(D)=−7.2° (c=1.5 MeOH); HPLC: (a) 70/30/0.05, 1.00 mL/min,282 nm, t_(R) 1.40 min, 99% pure; (b) 50/50/0.05, 2.0 mL/min, 276 nm,t_(R) 1.51 min, 96.1% pure.

(S,R)-(+)-1-p-Methoxyphenyl-2-(β-3′,5′-dihydroxyphenyl-β-oxy)ethylaminopropaneFumarate [(S,R)-2]

Prepared from (S)-8 and (R)-11 according to Procedure A to give 200 mg(51%). ¹H NMR (CD₃OD) δ 1.12 (d, 3H, J=6.0 Hz), 2.58-3.13 (m, 5H), 3.77(s, 3H), 4.62 (dd, 1H, J=3.6, 9.0 Hz), 6.15 (m, 1H), 6.30 (d, 2H, J=1.8Hz), 6.85 (d, 2H, J=8.7 Hz), 7.11 (d, 2H, J=8.7 Hz); ¹³C NMR (CD₃OD) δ18.2, 41.4, 54.1, 55.7, 56.5, 64.7, 103.0, 105.3, 115.1, 130.7, 131.3,145.9, 159.8, 160.0; UV (MeOH) λ_(max) □277 nm (∈ 3,150), 224 (3,310),205 (30,600); MS (ESI+) m/z (rel): 318 (100, M+H); [α]_(D)=+14.1°(c=0.95 MeOH); HPLC: (a) 70/30/0.05, 1.00 mL/min, 282 nm, t_(R) 1.42min, 97.7% pure; (b) 50/50/0.05, 2.0 mL/min, 276 nm, t_(R) 1.52 min,97.8% pure.

(R,R)-(+5-{2-[2-(4-Aminophenyl)-1-methylethylamino]-1-hydroxyethyl}-1,3-benzenediolFumarate [(R,R)-3]

Prepared from (R)-8 and (R)-12 according to Procedure A to give 88 mg(42%). ¹H NMR (CD₃OD) δ 1.23 (m, 3H), 2.70-3.24 (m, 4H), 3.54 (m, 1H),4.84 (dd, 1H, J=3.3, 9.6 Hz), 6.23 (t, 1H, J=2.4 Hz), 6.38 (d, 2H, J=2.1Hz), 6.75 (s, 2H, fumarate), 7.35 (dd, 4H, J=8.1, 21.0 Hz); ¹³C (CD₃OD)δ 15.5, 39.6, 52.7, 56.6, 70.3, 103.4, 105.3, 123.8, 132.0, 132.1,135.2, 137.5, 144.7, 160.0, 168.1; UV (MeOH) λ_(max) 284 nm (∈ 1,520),206 (21,700); MS (ESI+) m/z (rel): 303 (100, M+H); [α]_(D)=−6.8°(conc=1.0% MeOH); HPLC: (a) 80/20/0.05, 0.70 mL/min, 276 nm, t_(R) 2.07min, 95.5% pure; (b) 50/50/0.05, 1.0 mL/min, 282 nm, t_(R) 2.60, 97.16%pure.

(S,S)-(+)-5-{2-[2-(4-Aminophenyl)-1-methylethylamino]-1-hydroxyethyl}-1,3-benzenediolFumarate [(S,S)-3]

Prepared from (S)-8 and (S)-12 according to Procedure A to give 56 mg(25%). ¹H NMR (CD₃OD) δ 1.23 (m, 3H), 2.62-3.27 (m, 4H), 3.55 (m, 1H),4.74-4.88 (m, 1H), 6.22 (t, 1H, J=1.8 Hz), 6.37 (d, 2H, J=2.4 Hz), 6.75(s, 2H, fumarate), 7.32 (dd, 4H, J=8.7, 25.8 Hz); ¹³C NMR (CD₃OD) δ15.5, 39.6, 52.5, 56.7, 70.7, 103.4, 105.3, 123.3, 131.8, 132.0, 135.2,136.9, 144.7, 160.0, 168.1; UV (MeOH) λ_(max) 284 nm (∈ 1,720), 207(28,400); MS (ESI+) m/z (rel): 303 (100, M+H), 329 (20); [α]_(D)=+11.1°(conc=0.50% MeOH); HPLC: (a) 80/20/0.05, 0.7 mL/min, 276 nm, t_(R) 2.01min, <99% pure; (b) 50/50/0.05, 1.0 mL/min, 282 nm, t_(R) 2.50 min,99.4% pure.

(R,S)-(−)-5-{2-[2-(4-Aminophenyl)-1-methylethylamino]-1-hydroxyethyl}-1,3-benzenediolFumarate [(R,S)-3]

Prepared from (R)-8 and (S)-12 according to Procedure A to give 72 mg(35%). ¹H NMR (CD₃OD) δ□ 1.23 (m, 3H), 2.73-3.24 (m, 4H), 3.51 (m, 1H),4.80 (dd, 1H, J=2.7, 9.6 Hz), 6.22 (t, 1H, J=2.1 Hz), 6.36 (d, 2H, J=2.4Hz), 6.75 (s, 2H, fumarate), 7.32 (dd, 4H, J=8.4, 25.2 Hz); ¹³C NMR(CD₃OD) δ 16.1, 39.12, 5.16, 56.9, 70.4, 103.4, 105.3, 123.4, 132.0,132.0, 135.2, 136.8, 144.6, 160., 168.10; UV (MeOH) λ_(max) 284 nm (∈1,620), 205 (27,200); MS (ESI+) m/z (rel): 303 (100, M+H), 134 (14);[α]D=−7.5° (conc=0.50% MeOH); HPLC: (a) 80/20/0.05, 0.7 mL/min, 276 nm,t_(R) 2.08 min, 95.0% pure; (b) 50/50/0.05, 1.0 mL/min, 282 nm, t_(R)2.51 min, 97.4% pure.

(S,R)-(+)-5-{2-[2-(4-Aminophenyl)-1-methylethylamino]-1-hydroxyethyl}-1,3-benzenediolFumarate [(S,R)-3]

Prepared from (S)-8 and (R)-12 according to Procedure A to give 93 mg(42%). ¹H NMR (CD₃OD) δ□1.23 (d, 3H, J=6.3 Hz), 2.70-3.78 (m, 4H),3.42-3.62 (m, 1H), 4.80 (dd, 1H, J=3.0, 9.9 Hz), 6.22 (t, 1H, J=2.1 Hz),6.37 (d, 2H, J=2.1 Hz), 6.75 (s, 2H, fumarate), 7.33 (dd, 4H, J=8.4,26.7 Hz); ¹³C NMR (CD₃OD) δ 16.2, 39.1, 52.6, 56.9, 70.5, 103.4, 105.3,123.5, 132.1, 133.7, 135.2, 137.1, 144.7, 160.0, 168.1 UV (MeOH) λ_(max)284 nm (∈ 8,230), 207 (100,000); MS (ESI+) m/z (rel): 303 (100, M+H),134 (18); [α]_(D)=+11.4° (conc=0.50% MeOH); HPLC: (a) 70/30/0.05, 1.00mL/min, 280 nm, t_(R) 1.45 min, 99% pure; (b) 50/50/0.05, 1.0 mL/min,282 nm, t_(R) 2.63 min, 95.33% pure.

(R,R)-(−)-5-[1-Hydroxy-2-(1-methyl-2-phenylethylamino)ethyl]-1,3-benzenediolfumarate [(R,R)-4]

Prepared from (R)-8 and (R)-13 according to Procedure A to give 92 mg(26%). ¹H NMR (CD₃OD) δ□1.22 (m, 3H), 2.68-3.28 (m, 2H), 3.10-3.28 (m,2H), 3.53 (br-m, 1H), 4.75-4.80 (m, 1H), 6.24 (t, 1H, J=2.4 Hz), 6.38(d, 2H, J=2.1 Hz), □ 6.75 (s, 1H, fumarate), 7.22-7.33 (m, 5H); □¹³C NMR(CD₃OD) δ 15.5, 40.3, 56.9, 70.2, 103.4, 105.3, 128.3, 129.9, 130.3,135.2, 137.3, 144.6, 144.6, 159.9, 168.1; UV (MeOH) λ_(max) 277 nm (∈926), 204 (18,700); MS (APCI+) m/z 288 (100, M+H); [α]_(D)=−21.2°(conc=0.85% MeOH); HPLC: (a) 50/50/0.05, 1.00 mL/min, 282 nm; t_(R) 1.73min; 99% pure; (b) 50/50/0.05, 2.0 mL/min, 276 nm, t_(R) 1.46 min, 97.5%pure.

(S,S)-(+)-5-[1-Hydroxy-2-(1-methyl-2-phenylethylamino)ethyl]-1,3-benzenediolfumarate [(S,S)-4]

Prepared from (S)-8 and (S)-13 according to Procedure A to give 184 mg(51%). ¹H NMR (CD₃OD) δ□1.21 (m, 3H), 2.70-3.13 (m, 2H), 3.15-3.23 (m,2H), 3.54 (br-m, 1H), 4.79-4.86 (m, 1H), 6.24 (t, 1H, J=2.1 Hz), 6.39(t, 2H, J=2.7 Hz), 6.76 (s, 1H, fumarate), 7.22-7.32 (m, 5H); ¹³C NMR(CD₃OD) δ 15.5, 40.3, 56.9, 70.2, 103.4, 105.3, 128.3, 129.9, 130.3,135.1, 137.3, 144.6, 144.6, 159.9, 168.1 UV (MeOH) λ_(max) 278 nm (∈1,510), 207 (26,600); MS (APCI+) m/z 288 (100, M+H); [α]_(D)=+19.3°(conc=0.90% MeOH); HPLC: (a) 50/50/0.05, 1.00 mL/min, 282 nm, t_(R) 1.49min; 98.4% pure; (b) 50/50/0.05, 2.0 mL/min, 276 nm, t_(R) 1.35 min, 99%pure.

(R,S)-(+5-[1-Hydroxy-2-(1-methyl-2-phenylethylamino)ethyl]-1,3-benzenediolfumarate [(R,S)-4]

Prepared from (R)-8 and (S)-13 according to Procedure A to give 170 mg(45%). ¹H NMR (CD₃OD) δ 1.22 (m, 3H), 2.68-3.28 (m, 2H), 3.13-3.28 (m,2H), 3.53 (br-m, 1H), 4.76-4.80 (m, 1H), 6.23 (t, 1H, J=2.1 Hz), 6.37(t, 2H, J=3.0 Hz), 6.75 (s, 1H, fumarate), 7.24-7.37 (m, 5H); ¹³C NMR(CD₃OD) δ 16.3, 24.2, 39.8, 57.2, 70.5, 103.4, 105.3, 128.4, 130.0,130.4, 135.2, 137.4, 144.6, 160.1 UV (MeOH) λ_(max) 278 nm (δ 1,110),205 (31,000); MS (APCI+) m/z (rel): 288 (100, M+H); [α]_(D)=−6.9°(conc=0.85% MeOH); HPLC: (a) 50/50/0.05, 1.00 mL/min, 282 nm, t_(R) 1.53min, 99% pure; (b) 50/50/0.05, 2.0 mL/min, 276 nm, t_(R) 1.46 min, 98.5%pure.

(S,R)-(+)-5-[1-Hydroxy-2-(1-methyl-2-phenylethylamino)ethyl]-1,3-benzenediolfumarate [(S,R)-4]

Prepared from (S)-8 and (R)-13 according to Procedure A to give 212 mg(59%). ¹H NMR (CD₃OD) δ 1.22 (m, 3H), 2.72 (dd, 1H J=10.2, 13.2 Hz),3.11 (dd, 1H, J=10.2, 12.6 Hz), 3.18-3.27 (m, 2H), 3.48-3.61 (m, 1H),4.83 (dd, 1H, J=3.3, 9.9 Hz), 6.22 (t, 1H, J=2.4 Hz), 6.36 (d, 2H, J=2.4Hz), 6.75 (s, 1H, fumarate), 7.24-7.37 (m, 5H); ¹³C NMR (CD₃OD) δ 16.3,24.2, 39.8, 57.2, 70.5, 103.4, 105.3, 128.4, 130.0, 130.4, 135.2, 137.4,144.6, 160.1; UV (MeOH) λ_(max) 278 nm (∈ 1,680), 206 (35,500); MS(APCI+) m/z (rel): 288 (100, M+H), 270 (19, M−OH); [α]_(D)=+9.1°(conc=1.1%, MeOH); HPLC: (a) 50/50/0.05, 1.00 mL/min, 282 nm, t_(R) 1.51min, 99% pure; (b) 50/50/0.05, 2.0 mL/min, 276 nm, t_(R) 1.43 min, 99%pure.

(R,R)-(−)-5-{1-hydroxy-2-[1-methyl-2-(1-naphthyl)ethylamino]ethyl}-1,3-benzenediolfumarate [(R,R)-5]

Prepared from (R)-8 and (R)-14 according to Procedure A to give 135 mg(46%). ¹H NMR (CD₃OD) δ 1.18-1.23 (m, 3H), 3.16-3.34 (m, 1H, 2H),3.69-3.74 (m, 2H), 4.78-4.80 (m, 1H), 6.23 (t, 1H, J=2.4 Hz), 6.38 (m,2H), 7.41-7.61 (m, 4H), 7.83 (d, 1H, J=7.5 Hz), 7.90 (d, 1H, J=7.8 Hz),8.10 (m, 1H); ¹³C NMR (CD₃OD) δ 16.2, 37.2, 54.5, 56.1, 70.3, 103.4,105.3, 124.3, 126.5, 127.0, 127.7, 129.3, 130.1, 133.2, 135.2, 135.6,144.7, 160.1, 168.2; UV (MeOH) λ_(max) 282 nm (∈ 5,860), 224 (50,900),208 (35,500); MS (APCI+) m/z (rel): 338 (100, M+H), 169 (15, fragment);[α]_(D)=−20.4 (conc=0.50% MeOH); HPLC: (a) 60/40/0.05, 1.00 mL/min, 282nm, t_(R) 2.08 min, 95.7% pure; (b) 50/50/0.05, 1.5 mL/min, 282 nm,t_(R) 2.20 min, 99% pure.

(S,S)-(+)-5-{1-hydroxy-2-[1-methyl-2-(1-naphthyl)ethylamino]ethyl}-1,3-benzenediolfumarate [(S,S)-5]

Prepared from (S)-8 and (S)-14 according to Procedure A to give 118 mg(40%) ¹H NMR (CD₃OD) δ 1.13-1.17 (m, 3H), 3.14-3.26 (m, 1H, 2H),3.61-3.76 (m, 2H), 4.44-4.75 (m, 1H), 6.18 (t, 1H, J=2.4 Hz), 6.33 (m,2H), 7.36-7.52 (m, 4H), 7.77 (dd, 1H, J=1.8, 7.5 Hz), 7.84 (d, 1H, J=8.1Hz), 8.04 (t, 1H, J=8.4 Hz); ¹³C NMR (CD₃OD) δ 16.1, 37.1, 54.4, 56.0,70.3, 103.3, 105.3, 124.3, 126.5, 127.0, 127.7, 129.2, 130.0, 133.2,135.2, 135.7, 144.5, 160.0, 168.1; UV (MeOH) λ_(max) 282 nm (∈ 6,210),223 (56,400), 208 (42,700); MS (APCI+) m/z (rel): 338 (100, M+H), 169(8, fragment); [α]_(D)=+20.0° (conc=1.1% MeOH); HPLC: (a) 60/40/0.05,1.00 mL/min, 282 nm, t_(R) 2.35 min, 98.9% pure; (b) 50/50/0.05, 1.5mL/min, 282 nm, t_(R) 2.26 min, 97.2% pure.

(R,S)-(−)-5-{1-hydroxy-2-[1-methyl-2-(1-naphthyl)ethylamino]ethyl}-1,3-benzenediolfumarate [(R,S)-5]

Prepared from (R)-8 and (S)-14 according to Procedure A to give 114 mg(39%). ¹H NMR (CD₃OD) δ 1.08-1.11 (m, 3H), 3.02-3.24 (m, 1H, 2H),3.54-3.68 (m, 2H), 4.45-4.75 (m, 1H), 6.11 (t, 1H, J=1.8 Hz), 6.26 (m,2H), 6.63 (s, 2H fumarate), 7.28-7.48 (m, 4H), 7.70 (d, 1H, J=7.5 Hz),7.77 (d, 1H, J=7.8 Hz), 7.97 (t, 1H, J=7.8 Hz); ¹³C NMR (CD₃OD) δ 16.0,37.1, 52.5, 56.0, 70.4, 103.4, 105.3, 124.4, 126.5, 127.0, 127.6, 129.2,130.1, 133.1, 135.2, 135.5, 144.7, 159.9, 168.2; UV (MeOH) □λ_(max) 281nm (∈ 12,600), 224 (61,900), 204 (47,200); MS (APCI+) m/z (rel): 338(100, M+H), 190 (15, fragment); [α]_(D)=−11.3° (conc=0.85% MeOH); HPLC:(a) 60/40/0.05, 1.00 mL/min, 282 nm, t_(r) 2.30 min, 98.6% pure; (b)50/50/0.05, 1.5 mL/min, 282 nm, t_(R) 2.36 min, 99% pure.

(S,R)-(+)-5-{1-hydroxy-2-[1-methyl-2-(1-naphthyl)ethylamino]ethyl}-1,3-benzenediolfumarate [(S,R)-5]

Prepared from (S)-8 and (R)-14 according to Procedure A to give 123 mg(42%). ¹H NMR (CD₃OD) δ 1.18-1.22 (m, 3H), 3.10-3.28 (m, 1H, 2H),3.69-3.78 (m, 2H), 4.45-4.75 (m, 1H), 6.23 (t, 1H, J=2.1 Hz), 6.39 (m,2H), 6.73 (s, 2H fumarate), 7.39-7.59 (m, 4H), 7.80 (d, 1H, J=7.5 Hz),7.88 (d, 1H, J=7.8 Hz), 8.01 (t, 1H, J=9.0 Hz); ¹³C NMR (CD₃OD) δ 16.4,37.4, 52.5, 56.2, 70.6, 103.4, 105.3, 124.4, 126.5, 127.0, 129.3, 130.1,133.1, 133.4, 135.6, 136.3, 144.8, 160.0, 171.4; UV (MeOH) λ_(max) 282nm (607,740), 224 (70,900), 206 (55,800); MS (ESI+) m/z (rel): 338 (100,M+H); [α]_(D)=+15.5 (conc=1.0% MeOH) HPLC: (a) 60/40/0.05, 1.0 mL/min,282 nm, t_(R) 1.95, 95.7% pure; (b) 50/50/0.05, 1.5 mL/min, 282 nm,t_(R) 2.29 min, 95.7% pure.

(R,R)-(−)-5-[1-Hydroxy-2-(1-methylhexylamino)ethyl]-1,3-benzenediolFumarate [(R,R)-6]

Prepared from (R)-8 and (R)-15 according to Procedure A to give 45 mg(29%). ¹H NMR (CD₃OD) δ 0.920 (t, 3H, J=6.9 Hz), 1.30 (d, 3H, J=6.9 Hz),1.29-1.64 (m, 8H), 3.01-3.18 (m, 2H), 3.14-3.30 (m, 1H), 4.80 (dd, 1H,J=3.3, 9.6 Hz), 6.22 (t, 1H, J=2.1 Hz), 6.36 (d, 2H, J=2.4 Hz), 6.75 (s,1H, fumarate); ¹³C NMR (CD₃OD) δ 14.3, 16.0, 23.5, 26.2, 32.6, 34.2,52.1, 55.7, 70.2, 103.3, 105.3, 135.2, 144.7, 160.0, 168.0; UV (MeOH)λ_(max) 278 nm (∈ 931), 203 nm (20,100); MS (ESI+) m/z (rel): 268 (100,M+H); [α]_(D)=−8.8° (conc=1.1% MeOH); HPLC: (c) 70/30/0.1, 1.0 mL/min,276 nm, t_(R) 2.18 min, 96.6% pure; (b) 50/50/0.05, 1.0 mL/min, 279 nm,t_(R) 2.06 min, 98.9% pure.

(S,S)-(+)-5-[1-Hydroxy-2-(1-methylhexylamino)ethyl]-1,3-benzenediolFumarate [(S,S)-6]

Prepared from (S)-8 and (S)-15 according to Procedure A to give 96 mg(43%). ¹H NMR (CD₃OD) δ 0.923 (t, 3H, J=6.6 Hz); 1.31 (d, 3H, J=6.6 Hz),1.26-1.84 (m, 8H), 2.01-3.18 (m, 2H), 3.14-3.30 (m, 1H), 4.81 (dd, 1H,J=3.3, 9.6 Hz), 6.23 (t, 1H, J=2.4 Hz), 6.39 (d, 2H, J=2.1 Hz), 6.76 (s,1H, fumarate): ¹³C NMR (CD₃OD) δ 14.2, 16.0, 23.4, 26.3, 32.6, 34.1,52.1, 55.8, 70.2, 103.4, 105.3, 135.2, 144.7, 159.9, 168.2; UV (MeOH)λ_(max) 278 nm (∈ 1,340), 203 (28,800); MS (APCI+) m/z (rel): 268 (100,M+H); [α]_(D)=+10.8° (conc=0.50% MeOH); HPLC: (c) 70/30/0.1, 1.0 mL/min,276 nm, t_(R) 2.16 min, 97.0% pure; (b) 50/50/0.05, 1.0 mL/min, 279 nm,t_(R) 2.11 min, 99% pure.

(R,S)-(+5-[1-Hydroxy-2-(1-methylhexylamino)ethyl]-1,3-benzenediolFumarate [(R,S)-6]

Prepared from (R)-8 and (S)-15 according to Procedure A to give 83 mg(38%). ¹H NMR (CD₃OD) δ 0.924 (m, 3H); 1.32 (d, 3H, J=6.6 Hz), 1.26-1.84(m, 8H), 2.98-3.20 (m, 2H), 3.32-3.22 (m, 1H), 4.78 (dd, 1H, J=3.0, 9.9Hz), 6.23 (t, 1H, J=2.1 Hz), 6.37 (d, 2H, J=1.8 Hz), 6.76 (s, 1H,fumarate); ¹³C NMR (CD₃OD) 14.2, 16.4, 23.4, 26.2, 32.6, 33.5, 52.2,56.0, 70.4, 103.4, 105.3, 135.2, 144.7, 160.0, 168.1; UV (MeOH) λ_(max)276 nm (∈ 2,770), 203 (35,900); MS (APCI+) m/z (rel): 268 (100, M+H);[α]_(D)=−15.9° (conc=0.70% MeOH); HPLC: (c) 70/30/0.1, 1.0 mL/min, 276nm, t_(R) 2.16 min, 97.0% pure; (b) 50/50/0.05, 1.0 mL/min, 279 nm,t_(R) 2.07 min, 96.2% pure.

(S,R)-(+)-5-[1-Hydroxy-2-(1-methylhexylamino)ethyl]-1,3-benzenediolFumarate [(S,R)-6]

Prepared from (S)-8 and (R)-15 according to Procedure A to give 81 mg(38%). ¹H NMR (CD₃OD) δ 0.920 (t, 3H, J=6.3 Hz), 1.32 (d, 3H, J=6.9 Hz),1.30-1.77 (m, 8H), 2.99-3.17 (m, 2H), 3.23-3.26 (m, 1H), 4.76 (dd, 1H,J=3.0, 9.6 Hz), 6.22 (t, 1H, J=2.4 Hz), 6.36 (d, 2H, J=2.1 Hz), 6.75 (s,1H, fumarate); ¹³C NMR (CD₃OD) δ 14.2, 16.5, 23.5, 26.2, 32.6, 39.5,52.2, 56.0, 70.4, 103.4, 105.3, 135.2, 144.7, 160.0, 168.0; UV (MeOH)λ_(max) 278 nm (∈ 1,440), 204 (29,900); MS (APCI+) m/z (rel): 268 (100,M+H); [α]_(D)=+12.7° (conc=1.0% MeOH); HPLC: (c) 70/30/0.1, 1.0 mL/min,276 nm, t_(R) 2.16 min, 99% pure; (b) 50/50/0.05, 1.0 mL/min, 279 nm,t_(R) 2.02 min, 95.7% pure.

(R)-(−)-5-(1-Hydroxy-2-phenethylaminoethyl)-1,3-benzenediol fumarate[(R)-7]

Prepared from (R)-8 and 28 to give 37 mg (15%). ¹H NMR (CD₃OD) δ2.94-3.23 (m, 6H), 4.73 (dd, 1H, J=3.3, 9.9 Hz), 6.15 (t, 1H, J=2.4 Hz),6.29 (d, 2H, J=1.8 Hz), 7.19-7.28 (m, 5H), 6.69 (s, 1H); UV (MeOH)λ_(max) 278 nm (∈ 1,360), 205 (32,600); MS (APCI+) m/z (rel): 274 (100,M+H); [α]_(D)=−13.0° (conc=1.0% MeOH); HPLC: (a) 80/20/0.05, 1.00mL/min, 282 nm, t_(R) 1.47 min, 96.7% pure; (b) 50/50/0.05, 1.0 mL/min,272 nm, t_(R) 2.78 min, 95.1% pure.

(S)-(+)-5-(1-hydroxy-2-phenethylaminoethyl)-1,3-benzenediol fumarate[(S)-7]

Prepared from (S)-8 and 28 to give 51 mg (17%). ¹H NMR (CD₃OD)δ□2.87-3.21 (m, 6H), 4.68 (dd, 1H, J=3.6, 9.9 Hz), 6.10 (t, 1H, J=2.4Hz), 6.24 (d, 2H, J 2.1 Hz), 6.63 (s, 1H), 7.12-7.21 (m, 5H); UV (MeOH)λ_(max) 278 nm (∈ 1,280), 204 (33,700); MS (APCI+) m/z (rel): 274 (100,M+H); [α]_(D)=+14.64° (conc=1.1% MeOH); HPLC: (a) 80/20/0.05, 1.00mL/min, 282 nm, t_(R) 1.47 min, 98.6% pure; (b) 50/50/0.05, 1.0 mL/min,272 nm, t_(R) 2.74 min, 98.8% pure.

(R,R)-(−)-ethylfenoterol

¹H NMR: (300 MHz, CD₃OD): δ 0.950 (t, 3H, J=7.5 Hz), 1.67 (m, 2H),2.83-3.18 (m, 4H), 3.33-3.40 (m, 1H), 3.37 (s, 4H), 4.82 (m, 1H), 6.24(d, 1H, J=2.1 Hz), 6.37 (d, 2H, J=1.8 Hz), 6.73 (s, 2H, fum), 6.76 (d,2H, J=8.4 Hz), 7.05 (d, 2H, J=8.7 Hz) ppm. CMR: ¹³C (75 MHz, CD₃OD): δ9.43, 23.28, 36.56, 52.29, 62.16, 70.02, 103.4, 105.3, 116.7, 127.8,131.3, 136.5, 144.6, 157.6, 159.9, 172.3 ppm. UV: (Methanol), λ_(max)(∈): 206 nm (22,500), 223 (12,300), 278 (2,460). MS: (LCQ DUO ESIpositive ion mass spectrum) M/z (rel): 318 (100, M+H). HPLC 1: Column:Varian Sunfire C18 100×4.6; 70/30/0.1 water/acetonitrile/TFA; 1.0mL/min; Det: 278 nm; 2.76 min (fumarate, 6.99%), 3.57 min (90.11%);Purity: 97.1%. HPLC 2: Column: Chiralpak IA 250×10; 90/10/0.05acetonitrile/methanol/TFA; 2.0 mL/min; Det: 278 nm; 5.26 (RR isomer,92.37%), 7.11 min (fumarate, 5.02%); Purity 97.5%. Specific Rotation:[α]_(D)=−15.6 (free amine, 0.5% MeOH).

(R,S)-(−)-ethylfenoterol

¹H NMR: (300 MHz, CD₃OD): δ 0.972 (t, 3H, J=7.5 Hz), 1.70 (p, 2H, J=6.9Hz)), 2.86-3.22 (m, 4H), 3.32-3.37 (m, 1H), 3.34 (s, 4H), 4.82 (m, 1H),6.25 (t, 1H, J=2.1 Hz), 6.36 (d, 2H, J=1.8 Hz), 6.74 (s, 2H, fum), 6.77(d, 2H, J=8.4 Hz), 7.08 (d, 2H, J=8.7 Hz) ppm. CMR: ¹³C (75 MHz, CD₃OD):δ 9.820, 24.16, 36.48, 52.30, 62.32, 69.92, 103.3, 105.3, 116.8, 127.7,131.3, 136.1, 144.4, 157.6, 159.8, 171.3 ppm. UV: (Methanol), λ_(max)(s): 204 nm (26,900), 224 (11,500), 278 (2,320). MS: (LCQ DUO ESIpositive ion mass spectrum) M/z (rel): 318 (100, M+H). HPLC 1: Column:Varian Sunfire C18 100×4.6; 70/30/0.1 water/acetonitrile/TFA; 1.0mL/min; Det: 278 nm; 2.79 min (fumarate, 3.34%), 3.56 min (96.11%);Purity: 99.5% HPLC 2: Column: Chiralpak IA 250×10; 90/10/0.05acetonitrile/methanol/TFA; 2.0 mL/min; Det: 278 nm; 5.88 (RS isomer,97.08%), 7.12 min (fumarate, 2.92%); Purity>99%. Specific Rotation:[α]_(D)=−7.2 (free amine, 0.5% MeOH).

C₂₂H₂₅NO₄.0.5C₄H₄O₄

¹H NMR: (300 MHz, CD₃OD): δ 1.22 (t, 3H, J=6.6 Hz), 3.09-3.21 (m, 3H),3.59-3.69 (m, 2H), 3.99 (s, 3H), 4.74-4.83 (m, 1H), 6.23 (t, 1H, J=2.4Hz), 6.37 (dd, 2H, J=2.4, 5.7 Hz), 6.74 (s, 1H), 6.86 (d, 1H, J=7.8 Hz),7.32 (d, 1H, J=7.8 Hz), 7.48 (t, 1H, J=6.9 Hz), 7.56 (t, 1H, J=6.9 Hz),8.02 (dd, 1H, J=8.4, 12.0 Hz), 8.27 (d, 1H, J=8.7 Hz) ppm. CMR: ¹³C (75MHz, CD₃OD): δ 15.78, 36.66, 52.39, 55.96, 70.20, 103.4, 104.5, 105.3,123.8, 124.3, 124.9, 126.2, 127.4, 128.1, 129.5, 133.8, 135.2, 144.6,156.6, 160.0, 168.3 ppm. UV: (Methanol), λ_(max) (∈): 298 nm (4,970),286 (9,920), 234 (22,600), 210 (42,500). MS: (LCQ DUO ESI positive ionmass spectrum) M/z (rel): 368 (100, M+H). Specific Rotation:[α]_(D)=−28.8 (Free Amine; 0.5% MeOH).

C₂₂H₂₅NO₄.0.5C₄H₄O₄

¹H NMR: (300 MHz, CD₃OD): δ 1.20 (t, 3H, J=6.6 Hz), 3.07-3.21 (m, 3H),3.52-3.75 (m, 2H), 3.97 (s, 3H), 4.69-4.83 (m, 1H), 6.24 (t, 1H, J=2.1Hz), 6.39 (dd, 2H, J=2.4, 5.4 Hz), 6.74 (s, 1H), 6.84 (d, 1H, J=7.8 Hz),7.31 (d, 1H, J=8.1 Hz), 7.48 (t, 1H, J=6.9 Hz), 7.56 (t, 1H, J=6.9 Hz),8.01 (dd, 1H, J=8.4, 13.5 Hz), 8.27 (d, 1H, J=7.8 Hz) ppm. CMR: ¹³C (75MHz, CD₃OD): δ 15.77, 36.64, 52.37, 55.94, 70.46, 103.4, 104.5, 105.3,123.8, 124.3, 124.9, 126.2, 127.4, 128.1, 129.4, 133.8, 135.5, 144.7,156.6, 160.0, 169.0 ppm. UV:(Methanol), λ_(max) (∈): 298 nm (5,430), 286(5,710), 233 (25,100), 210 (43,200). MS: (LCQ DUO ESI positive ion massspectrum) M/z (rel): 368 (100, M+H). Specific Rotation: [α]_(D)=−15.8(Free Amine; 0.5% MeOH).

A step in the synthesis of the 4 stereoisomers of 1-6 was the couplingof the epoxide formed from either (R)- or(S)-3′,5′-dibenzyloxyphenylbromohydrin with the (R)- or (S)-enantiomerof the appropriate benzyl-protected 2-amino-3-benzylpropane (1-5) or the(R)- or (S)-enantiomer of N-benzyl-2-aminoheptane (6), Scheme I.

The synthesis of (R)-7 and (S)-7 was accomplished using2-phenethylamine, Scheme II. This approach was similar to the onedeveloped by Trofast et al. (Chirality 3: 443-450, 1991) for thesynthesis of the stereoisomers of formoterol, compound 47, FIG. 6. Theresulting compounds were then deprotected by hydrogenation over Pd/C andpurified as the fumarate salts.

The chiral building blocks used in the syntheses were produced usingScheme III. The (R)- and (S)-3′,5′-dibenzyloxyphenyl-bromohydrinenantiomers were obtained by the enantiospecific reduction of3,5-dibenzyloxyα-bromoacetophenone using boron-methyl sulfide complex(BH₃SCH₃) and either (1R,2S)- or (1S,2R)-cis-1-amino-2-indanol. Therequired (R)- and (S)-2-benzylaminopropanes were prepared byenantioselective crystallization of the rac-2-benzylaminopropanes usingeither (R)- or (S)-mandelic acid as the counter ion.

Example 6 Binding Affinities of Exemplary Fenoterol Analogues for β1 andβ2 Adrenergic Receptors

This example demonstrates that fenoterol analogues have an equivalent ifnot greater binding affinity for β2-adrenergic receptors than fenoterol.

Compounds were tested up to three times each to determine their bindingaffinities at the γ_(□)- and β₂-adrenergic receptors. Competition curveswith standard and unknown compounds included at least six concentrations(in triplicate). For each compound, graphs were prepared containingindividual competition curves obtained for that test compound. IC₅₀values and Hill coefficients were calculated using GraphPad Prism®software. K_(i) values were calculated using the Cheng-Prusofftransformation (Biochem Pharmacol 22: 3099-3108, 1973). In eachexperiment, a standard compound was simultaneously run on the 96-wellplate. If the standard compound did not have an IC₅₀ value close to theestablished average for that compound, the entire experiment wasdiscarded and repeated again.

β₁-adrenergic receptor binding was done on rat cortical membranefollowing a previously described procedure (Beer et al., Biochem.Pharmacol. 37: 1145-1151, 1988). In brief, male Sprague-Dawley ratsweighing 250-350 g were decapitated and their brains quickly removed.The cerebral cortices were dissected on ice, weighed and promptlytransferred to a 50 ml test tube containing approximately 30 ml of 50 mMTris-HCl, pH 7.8 (at room temperature). The tissues were homogenizedwith a polytron and centrifuged at 20,000×g for 12 min at 4° C. Thepellet was washed again in the same manner and resuspended at aconcentration of 20 mg (original wet wt) per 1 ml in the assay buffer(20 mM Tris-HCl, 10 mM MgCl₂, 1 mM EDTA, 0.1 mM ascorbic acid at pH7.8). To block the β₂ sites present in the cortical membranepreparation, 30 nM ICI 118-551 was also added to the assay buffer. Towells containing 100 μl of the test drug and 100 μl of [³H]CGP-12177(1.4 nM final concentration), 0.8 ml of tissue homogenate was added.After 2 hours at 25° C., the incubation was terminated by rapidfiltration. Nonspecific binding was determined by 10 μM propranolol.

HEK 293 cells stability transfected with cDNA encoding human μ₂-AR(provided by Dr. Brian Kobilka, Stanford Medical Center, Palo Alto,Calif.) were grown in Dulbecco's Modified Eagle Medium (DMEM) containing10% fetal bovine serum (FBS), 0.05% penicillin-streptomycin, and 400μg/ml G418 as previously described (Pauwels et al., Biochem. Pharmacol.42: 1683-1689, 1991). The cells were scraped from the 150×25 mm platesand centrifuged at 500×g for 5 minutes. The pellet was homogenized in 50mM Tris-HCl, pH 7.7, with a Polytron, centrifuged at 27,000×g, andresuspended in the same buffer. The latter process was repeated, and thepellet was resuspended in 25 mM Tris-HCl containing 120 mM NaCl, 5.4 mMKCl, 1.8 mM CaCl₂, 0.8 mM MgCl₂, and 5 mM glucose, pH 7.4. The bindingassays contained 0.3 nM [³H]CGP-12177 in a volume of 1.0 ml. Nonspecificbinding was determined by 1 μM propranolol.

According to the above-described methods, binding affinities, expressedas K_(i) values, were determined using membranes obtained from a HEK 293cell line stably transfected with cDNA encoding human β₂-AR (Pauwels etal., Biochem. Pharmacol. 42: 1683-1689, 1991) with [³H]CGP-12177 as themarker ligand. The resulting IC₅₀ values and Hill coefficients werecalculated for each test compound using GraphPad Prism® software andK_(i) values were calculated using the Cheng-Prusoff transformation(Biochem Pharmacol 22: 3099-3108, 1973):

K _(i) =IC ₅₀/(1+L/K _(d))+  Eqn. 1.

Where: L is the concentration of [³H]CGP-12177 and K_(d) is the bindingaffinity of the [³H]CGP-12177. Each test compounds was assayed threetimes.

The relative binding affinities to the β₂-AR for the stereoisomers ofcompounds 1-4 and 6 were R,R>R,S>S,R≈S,S (FIG. 5; Table 1, below). Thisstereoselectivity is consistent with the previously reported potenciesof the formoterol stereoisomers (Trofast et al., Chiralty 3: 443-450,1991) and results from binding studies with the isoproterenol derivativePTFAM, compound 48, FIG. 6 (Eimerl et al., Biochem. Pharmacol. 36:3523-3527, 1987). With compound 5, no significant difference was foundbetween the K_(i) values of the R,R and R,S isomers, thus the order wasR,R=R,S>S,R>S,S. The K_(i) value for (R)-7 was greater than that of(S)-7, which is consistent with the established enantioselective bindingpreference for β₂-ARs with the R-configuration at the stereogenic centercontaining the n-OH moiety, c.f. (Eimerl et al., Biochem. Pharmacol. 36:3523-3527, 1987; Wieland et al., Proc. Natl. Acad. Sci. USA 93:9276-9281, 1996; Kikkawa et al., Mol. Pharmacol. 53: 128-134, 1998; andZuurmond et al., Mol. Pharmacol. 56: 909-916, 1999).

TABLE 1 The binding affinities to the β₂-AR of the compounds synthesizedin this study calculated as K_(i) ± SEM (nM), n = 3. Comparison of β₁-and β₂ adrenergic binding affinity of fenoterol isomers Compound K_(i)β₁ K_(i) β₂ K_(i) β₁/K_(i) β₂ (R,R)-1 14750 ± 2510 345 ± 34 43 (R,S)-118910 ± 2367 3695 ± 246 5 (S,R)-1 >100,000 10330 ± 1406 NC(S,S)-1 >100,000 27749 ± 6816 NC (R,R)-2 21992 ± 3096 474 ± 35 46(R,S)-2 30747 ± 6499 1930 ± 135 16 (S,R)-2 33378 ± 9170 5269 ± 509 6(S,S)-2 >100,000 15881 ± 2723 NC (R,R)-3 24956 ± 2100 2934 ± 168 9(R,S)-3 31324 ± 3485 7937 ± 397 4 (S,R)-3 77491 ± 3583 23125 ± 2093 3(S,S)-3 31440 ± 1681 28624 ± 906  1 (R,R)-4 17218 ± 1270 1864 ± 175 9(R,S)-4 33047 ± 2779 6035 ± 434 4 (S,R)-4 >100,000 30773 ± 3259 NC(S,S)-4 >100,000 28749 ± 1811 NC (R,R)-5 3349 ± 125 241 ± 38 14 (R,S)-515791 ± 6269 341 ± 23 46 (S,R)-5 34715 ± 9092 1784 ± 148 19(S,S)-5 >100,000 2535 ± 209 NC (R,R)-6 10185 ± 499  9275 ± 902 1(R,S)-6 >100,000 31440 ± 1681 NC (S,R)-6 61295 ± 5821 >100,000 NC(S,S)-6 52609 ± 1434 56420 ± 5186 1 (R)-7 42466 ± 3466 10466 ± 1461 4(S)-7 52178 ± 3006 20562 ± 3721 3

When just the R,R isomers were compared, (R,R)-5 had the highestrelative affinity of the tested compounds, although the differencebetween (R,R)-5 and (R,R)-1 did not reach statistical significance,Table 1. The only other (R,R) stereoisomer with sub-micromolar affinitywas (R,R)-2, which had a significantly lower binding affinity than(R,R)-5, p=0.0051, and (R,R)-1, p=0.0291, although the mean K, value for(R,R)-2 is only 23% greater than that of (R,R)-1. The minimal effect oftransforming the p-OH moiety into a methyl ether is consistent withprevious data from Schirrmacher et al. (Bioorg. Med. Chem. Lett. 13:2687-92, 2003). In the previous study, rac-1 was converted into a[¹⁸F]-fluoroethoxy ether without significant loss of in vitro activityand it was concluded that, within the accuracy of the experimentalmeasurements, the derivatization did not change the binding affinity ofthe rac-1 to the β₂-AR.

Binding affinities, expressed as K_(i) values, for the β₁-AR weredetermined using rat cortical membranes with [³H]-CGP-12177 as themarker ligand (Beer et al., Biochem. Pharmacol. 37: 1145-1151, 1988.).The calculated K_(i) for (R,R)-5 was 3,349 nM and the binding affinitiesfor the all of the remaining test compounds were >10,000 nM, Table 1.Unlike the data from the β₂-AR binding studies, there was no clear trendwhich could be associated with the stereochemistry of the compounds.

The relative selectivity of the compounds for the β₂-AR and β₁-AR wasdetermined using the ratio K_(i) β₁/K_(i) β₂, Table 1. Of particularinterest were the ratios for the four compounds with sub-micromolaraffinity for the β₂-AR, (R,R)-1, (R,R)-2, (R,R)-5 and (RA-5, which were46, 43, 14 and 46, respectively. The results for (R,R)-1 and (R,R)-2 areconsistent with previously reported K_(i) β₁/K_(i) β₂ ratio of 53 forthe β₂-AR-selective agonist (R,R)-TA-2005, compound 49, FIG. 6.

The observed loss of β₂-AR selectivity for (R,R)-5 was unexpected as wasthe 3-fold increase in selectivity displayed by (R,S)-5 relative to(R,R)-5. Previous studies with the stereoisomers of 47 indicated thatboth the (R,R)- and (R,S)-isomers had a high degree of selectivity forthe β₂-AR, relative to the β₁-AR, with the selectivity of the(R,R)-isomer greater than that of the (R,S)-isomer (Trofast et al.,Chirality 3: 443-450, 1991). This is the case for compounds 1 and 2, butreversed for 5. It is also interesting to note that (S,R)-5 had asimilar selectivity (19-fold) and its affinity for the β₂-AR was only7-fold weaker than (R,R)-5, 1783 nM and 241 nM, respectively.

These studies demonstrate that (R,R)- or (R,S)-naphthyl fenoterolanalogues have a higher binding affinity for β2-adrenergic receptorsthan any isoform of fenoterol. The (R,R)-methyoxy fenoterol analogue hasa similar K_(i) for the β2-adrenergic receptor as (R,R)-fenoterol. Thus,such analogues are viable candidates for β2-adrenergic receptor agonistsand can likely be used to treat disorders that are presently treatedwith commercially available (±)-fenoterol.

Example 7 Cardiomyocyte Contractility Studies with Fenoterol Analogues

This example illustrates the pharmacological activities of the compoundswith sub-micromolar affinity for the □β₂-AR and (R,S)-1 and (S,S)-1.

In these studies, the contraction amplitude indexed by electrical pacinginduced shortening of cell length was measured in single ventricularmyocytes before and after exposure to a single dose of the testcompounds. Contractile response to the agonist was expressed as apercentage of the basal contractility and the specificity of the agonisttowards β₂-adrenergic receptor was determined by the inhibitory effectof ICI 118,551 (10⁻⁷ mol/L; Tocris Cookson Ltd., Bristol, U.K.), aselective □β₂-AR antagonist.

All of the compounds tested, except for (S,S)-1, produced a significantcontractile response which was blocked by ICI 118,551, while (S,S)-1 hadno observed pharmacological effect, FIG. 7. These results wereconsistent with the results from the previous study (Beigi et al.,Chirality, 18: 822-827, 2006) and with the observations that for agonistactivity at the β₂-AR an R-configuration is preferred at the stereogeniccenter containing the β-OH moiety, c.f. (Eimerl et al., Biochem.Pharmacol. 36: 3523-3527, 1987; Wieland et al., Proc. Natl. Acad. Sci.USA 93: 9276-9281, 1996; Kikkawa et al., Mol. Pharmacol. 53: 128-134,1998; and Zuurmond et al., Mol. Pharmacol. 56: 909-916, 1999). It is ofinterest to note that maximum effect was elicited with 0.1 μM (R,R)-5and (R,S)-5 while the other active compounds required 0.5 μMconcentrations. In addition, while the equivalent activities of (R,R)-5and (R,S)-5 were suggested by the binding data, the observed activity of(R,S)-1 was unexpected as previous studies of the stereoisomers of 47(Trofast et al., Chiralty 3: 443-450, 1991) and 48 (Eimerl et al.,Biochem. Pharmacol. 36: 3523-3527, 1987) indicated that the agonistactivities of the (R,R)-isomers were significantly greater than theactivities of the corresponding (R,S)-isomers.

Example 8 Comparative Molecular Field Analysis

This example illustrates the use of Comparative Molecular Field Analysis(CoMFA) to analyze the disclosed compounds.

The disclosed compounds were analyzed using Comparative Molecular FieldAnalysis, a 3D QSAR technique applicable to the analysis of the relativeactivities of stereoisomers and/or enantiomers at a selected target.

CoMFA was performed as implemented in SYBYL 7.2. (TRIPOS Inc., St.Louis, Mo.). Molecular models of all derivatives were prepared inHyperChem v. 6.03 (HyperCube Inc., Gainesville, Fla.) using ModelBuildprocedure to ensure the same conformation of the scaffold. Models wereextracted to SYBYL and the partial atomic charges (Gasteiger-Huckeltype) were calculated. Ligand models were aligned using as a commonsubstructure of the two asymmetric carbon atoms in the core of thefenoterol molecule (—C*—CH₂—NH—C*—CH₂—). Two types of molecular fields(steric and electrostatic) were sampled on the grid (2 Å spacing)lattice surrounding each structure. Distance-dependent dielectricconstant was used in electrostatic calculations and energetic cutoffs of30 kcal/mol for both the steric and the electrostatic energies were set.

The Partial Least Square correlation procedure applied for resultantdatabase extracted four statistically significant components and thefollowing validation parameters were obtained for the best solution:R²=0.920, F (4,21)=60.380, standard error of estimate=0.223,cross-validated (leave-one-out) R²=0.847. In general, electrostaticfields accounts for 48.1% of explained variance and steric fieldsaccounts for 51.9%. The resulting 3D QSAR model shows good statisticalcorrelation with experimental data, R²=0.920 and F=60.380, and goodprediction power as indicated by the cross-validated R² value (Q²)=0.847and the standard error of prediction (SEP)=0.309, Table 2.

TABLE 2 The pK_(d) predicted by the CoMFA model. Derivative pKd MeasuredpKd Predicted (R,R)-1 6.46 5.84 (R,S)-1 5.43 5.48 (S,R)-1 4.99 5.02(S,S)-1 4.56 4.66 (R,R)-2 6.32 6.17 (R,S)-2 5.71 5.80 (S,R)-2 5.28 5.34(S,S)-2 4.80 4.99 (R,R)-3 5.53 5.57 (R,S)-3 5.10 5.21 (S,R)-3 4.64 4.75(S,S)-3 4.54 4.39 (R,R)-4 5.73 5.58 (R,S)-4 5.22 5.25 (S,R)-4 4.51 4.75(S,S)-4 4.54 4.43 (R,R)-5 6.62 6.72 (R,S)-5 6.47 6.36 (S,R)-5 5.75 5.90(S,S)-5 5.60 5.54 (R,R)-6 5.03 5.01 (R,S)-6 4.50 4.66 (S,R)-6 4.00 4.19(S,S)-6 4.25 3.84 (R)-7 4.98 5.33 (S)-7 4.69 4.51

In the first stage, the model was used to identify the regionsresponsible for the discrimination between the stereoisomers. The CoMFAprocedure produced several distinct asymmetric regions located in closeproximity of each chiral center. The first chiral center (carrying the βhydroxyl group) is surrounded by an electropositive region behind themolecule. An electropositive region can be associated with hydrogen bondformation and indicates favorable donor properties or unfavorableacceptor properties of the pseudoreceptor. In this case, the location ofthe electropositive field indicates that the orientation of the β-OHmoiety behind the plane of the model (the S configuration at the chiralcenter) would hinder H bond formation with the receptor. Theelectropositive region is closely associated with a steric unfavorableregion behind the first chiral center. This is an additional indicationthat the model demonstrates a preference for the β-hydroxyl group in theR configuration. The preference for the R configuration at this centeris consistent with previous models and experimental data, whichdemonstrated that the R configuration is favored for functional activityat β-AR receptors (c.f., Eimerl et al., Biochem. Pharmacol. 36:3523-3527, 1987; Wieland et al., Proc. Natl. Acad. Sci. USA 93:9276-9281, 1996; Kikkawa et al., Mol. Pharmacol. 53: 128-134, 1998; andZuurmond et al., Mol. Pharmacol. 56: 909-916, 1999).

The CoMFA model also demonstrated the effect of the second chiralcenter. The preferred configuration can be derived from the bindingdata, where for compounds 1-4 and 6 the (R,R)-isomers had the higheraffinities relative to their respective (R,S)-isomers, while the K_(i)values for (R,R)-5 and (R,S)-5 were equivalent, Table 1. Thus, in thismodel, the more active isomers are those with the methyl moiety on thestereogenic center on the aminoalkyl portion of the molecules pointingout of the plane of the figure of the CoMFA model. This is depicted by asteric disfavoring region behind the second chiral center of themolecule, and indicates a preference for the R configuration at thissite.

In this study, only the aminoalkyl portion of the fenoterol molecule wasaltered and, therefore, the key CoMFA regions are associated with thisaspect of the molecule. In the resulting analysis, all four interactingregions were identified in the proximity of the aromatic moiety and allcan be used to generate hypotheses concerning the mode of binding actionof the studied derivatives.

In the model, the large electropositive region encompassing the areaclose to the —OH or OCH₃ substituents represents H-bond donor propertiesof the pseudoreceptor to these moieties. These interactions areresponsible for the relatively higher binding affinities of theO-derivatives, compounds 1 and 2, relative to compounds 3 and 4, in thelatter compound the p-amino substituent should be positively chargedunder the experimental conditions.

A large electronegative region and another electropositive region, bothlocated parallel on two sides of the aromatic system most likelyrepresent π-π or π-hydrogen bond interactions between the β₂-AR andelectron-rich aromatic moieties, such as the naphthyl ring. This isconsistent with the increased affinity of compounds 1, 2 and 5 relativeto the other compounds examined in this study. The role of thisinteraction is suggested by the observation that the K_(i) values for(R,R)-5 and (R,S)-5 were equivalent to (R,R)-1 and (R,R)-2, Table 1.

Two steric regions are located close to the electrostatic regions andone favors and the other disfavors bulkiness in the respective areas.This indicates that the binding of the aminoalkyl portions of themolecules are also sterically restricted.

The binding of agonists and antagonists to the β₂-AR has been studiedusing site-directed mutagenesis and molecular modeling techniques(Eimerl et al., Biochem. Pharmacol. 36: 3523-3527, 1987; Wieland et al.,Proc. Natl. Acad. Sci. USA 93: 9276-9281, 1996; Kikkawa et al., Mol.Pharmacol. 53: 128-134, 1998; Zuurmond et al., Mol. Pharmacol. 56:909-916, 1999; Kontoyianni et al., J. Med. Chem. 39: 4406-4420, 1996;Furse et al., J. Med. Chem. 46: 4450-4462, 2003; and Swaminath et al. JBiol. Chem. 279: 686-691, 2004). There is general agreement that thebinding of the “catechol” portion of an agonist occurs within a bindingarea created by the transmembrane (TM) helices identified as TM3, TM5and TM6. The binding process is a sequential event that producesconformational changes leading to G-protein activation (Furse et al., J.Med. Chem. 46: 4450-4462, 2003). A key aspect in this process is theinteraction of the hydroxyl moiety on the chiral carbon of the agonistwith the Asn-293 residue in TM6, and for this interaction anR-configuration is preferable at the chiral carbon (Eimerl et al.,Biochem. Pharmacol. 36: 3523-3527, 1987; Kikkawa et al., Mol. Pharmacol.53: 128-134, 1998; and Swaminath et al. J. Biol. Chem. 279: 686-691,2004). Since the “catechol” portion of the fenoterol molecule was notaltered in this study, it follows that in the CoMFA model, anR-configuration at the first stereogenic center is preferred in moststable complexes.

The majority of the binding and functional studies of β₂-AR agonistshave been conducted with small N-alkyl substituents such as methyl,isopropyl and t-butyl, c.f. (Kontoyianni et al., J. Med. Chem. 39:4406-4420, 1996). However, while these compounds are active at theβ₂-AR, they are not subtype selective. This is illustrated by the K_(i)β₁/K_(i) β₂ ratios determined for compounds 49, 50 and 51 (FIG. 6) whichwere 53, 1.7 and 1.3, respectively (Kikkawa, et al. Mol. Pharmacol. 53:128-134, 1998)). The removal of the p-methoxyphenyl moiety not onlyreduced the selectivity, but also the affinities as the respectiveβ₂K_(i) values were 12 nM, 170 nM and 6300 nM (Kikkawa, et al. Mol.Pharmacol. 53: 128-134, 1998).

The role that aminoalkyl substituents play in β₂-AR selectivity has beeninvestigated using site-directed mutagenesis and molecular modelingtechniques (Kikkawa, et al. Mol. Pharmacol. 53: 128-134, 1998); Furse etal., J. Med. Chem. 46: 4450-4462, 2003; and Swaminath et al. J. Biol.Chem. 279: 686-691, 2004). Using (R,R)-49 as the model ligand, Kikkawa,et al. determined that hydrogen bond formation between the p-methoxyoxygen on compound 49 and the hydroxyl group of tyrosine 308 (Y308)located in the extracellular end of TM7 was the source of the β₂-ARselectivity (Mol. Pharmacol. 53: 128-134, 1998).

Furse and Lybrand developed a de novo model of the β₂-AR andinvestigated molecular complexes of several ligands (agonist andantagonist) with this subtype (J. Med. Chem. 46: 4450-4462, 2003). Amongthe structures investigated, (R,R)-49 has the same aminoalkylsubstituent as the compound 2. Examination of the (R,R)-49/β₂-AR complexrevealed that the p-methoxy group oxygen of (R,R)-49 formed a hydrogenbond with the hydroxy group of Y308, which supports the model proposedby Kikkawa, et al. (Mol. Pharmacol. 53: 128-134, 1998). The distancebetween the two oxygen atoms bonded in the model was 3.22 Å. However,the methoxy moiety of the ligand was also located in close proximity tothree other polar residues, histidine 296 (H296) in TM6, tryptophan 109(W109) in TM3 and asparagine 312 (N312) in TM7, each of which caninteract with an aromatic group on the aminoalkyl portion of (R,R)-49.

In the Furse and Lybrand model, the distance between the oxygen atom ofthe ligand and the hydrogen atom of H296 was 5.88 Å and H296 wasproposed as an alternative hydrogen bond donor for interaction with themethoxy group of (R,R)-49. Since Y308 and H296 are found only in β₂-AR,the corresponding residues found in the β₁-AR are F359 and K347,respectively, the interaction with H296 and Y308 has been proposed asthe source of β₁/β₂ selectivity (Furse et al., J. Med. Chem. 46:4450-4462, 2003).

Since the previous studies of β₁/β₂ selectivity utilized (R,R)-49, thesubtype selectivity of the (R,R)-stereoisomers of the compoundssynthesized in our study were compared to the subtype selectivity of(R,R)-49. The data from this study suggest that hydrogen bond formationbetween Y308 and/or H296 and the oxygen atom on the p-substituent of theagonist is involved in β₂-AR selectivity. The interaction is possiblewith (R,R)-1 and (R,R)-2 and the K_(i) β₁/K_(i) β₂ ratios for thesecompounds are 43 and 46, respectively, which are comparable to the K_(i)β₁/K_(i) β₂ ratio of 53 determined for (R,R)-49. The K_(i) β₁/K_(i) β₂ratios for compounds 3, 4, 6 and 7 were <10 and reflect the fact thatthey do not have the ability to form hydrogen bonds with Y308 or H296.The hydrogen bonding interactions were also suggested by the CoMFA modelidentifying a large electropositive region surrounding the area close tothe —OH or —OCH₃ substituents, representing hydrogen-bond donorproperties of the pseudoreceptor.

The data from this study also suggest that an aromatic moiety on theaminoalkyl portion of the compound contributes to K_(i) and subtypeselectivity, even if the aromatic moiety is unable to form a hydrogenbond with the receptor. This is demonstrated by the comparison of theK_(i)β₂ values for the (R,R)-isomers of compounds 1-5 which were <3,000nM with K_(i)β₂ value of (R,R)-6 which was 9,000 nM and the K_(i)β₁/K_(i) β₂ ratios which were >9 for 1-5 while compound 6 displayed nosubtype selectivity, Table 1. One possible mechanism to explain the datais π-hydrogen bond formation. The cloud of π-electrons of aromatic ringscan act as hydrogen bond acceptors, although it has been estimated thatthe interaction would be about half as strong as a normal hydrogen bond(Levitt and Perutz J. Mol. Biol. 201: 751-754, 1998). The higheraffinity and subtype selectivity for (R,R)-5 relative to (R,R)-3 and(R,R)-4 or (R)-7 is consistent with the greater π electron distributionin the napthyl ring relative to the other aromatic rings.

The CoMFA model also identified a large electronegative region andanother electropositive region, both located parallel to the aromaticsystem, which are most likely associated with π-π or π-hydrogen bondinteractions between the β₂-AR and electron-rich aromatic moieties, suchas the naphthyl ring. Using the model developed by Furse and Lybrandwith (R,R)-49 as the interacting ligand, Y308, H296, W109 and N312 wereidentified as possible sources of π-π and/or π-hydrogen bondinteractions. In the β₂-AR model, the estimated distances between thep-methoxy moiety on (R,R)-49 and W109 and N312 were 4.80 Å and 3.45 Å,respectively. Since W109 and N312 are fully conserved in all β-ARsubtypes, the interactions suggested by the CoMFA model may representthe source of the increase affinities for (R,R)-1, (R,R)-2 and (R,R)-5,relative to the other (R,R)-isomers, but not the observed β₁/β₂selectivity.

The data from this study and the resulting CoMFA model indicate that thebinding process of the tested compounds with the β₂-AR includes theinteraction of the chiral center on the aminoalkyl portion of theagonist with a sterically restricted site on the receptor. The existenceof a sterically restricted site has been previously suggested from thedata obtained in the development of 3D models for agonist and antagonistcomplexes with the β₂-AR (Kobilka, Mol. Pharm. 65: 1060-1062, 2004). Forexample, (R,R)-49 and similar compounds with substituents larger than amethyl group at the stereogenic center on the aminoalkyl portion weresuggested to produce significant steric interactions that wouldunfavorably affect the ligand-receptor complexes.

The binding of an agonist to the β₂-AR has been described as a multistepinterrelated process, in which sequential interactions between theagonist and receptor produce corresponding conformational changes(Kobilka, Mol. Pharm. 65: 1060-1062, 2004). The CoMFA model reflects thefinal agonist/β₂-AR complex and, in order to discern the effect of thesteric restricted site, it is necessary to consider the effect thatinteraction with this site has on the outcome of the binding process. Adetailed description of the present CoMFA model is disclosed in Jozwiaket al. (J. Med. Chem., 50 (12): 2903-2915, 2007) which is herebyincorporated by reference in its entirety.

If one assumes that the interaction of the “catechol” portion of theagonist with the binding area created by TM3, TM5 and TM6 (the firstbinding area), then these interactions will fix the position of theaminoalkyl portion of the agonist relative to the steric restrictedsite, and perhaps even create this site. In the CoMFA model, the stericrestrictions at the site force the methyl moiety at the chiral center ofthe aminoalkyl portion to point out of the plane of the model.

Due to the free rotation about the N-atom, the configuration at thechiral center bearing the methyl moiety may likely not affect theability of the molecule to minimize the interaction with the stericrestricted site. However, in the minimum energy conformation, e.g., withthe methyl group pointing out of the plane of the CoMFA model, theorientation of the remaining segment of the aminoalkyl portion relativeto the second binding area would be affected by the stereochemistry.Indeed, R and S configurations would produce mirror image relationshipsto the second binding area. This situation is illustrated in FIG. 5where the catechol, first chiral center and the methyl moieties of(R,R)-5 and (R,S)-5 have been overlaid upon each other.

The studies elucidating the source of β₂-AR selectivity have primarilyutilized (R,R)-49 and one previous study of the effect of chirality onsubtype selectivity reported that (R,R)-47 had a higher β₂-ARselectivity than (R,S)-47 (Trofast et al., Chiralty 3: 443-450, 1991).Thus, the observed equivalent affinities and functional activities of(R,R)-5 and (R,S)-5 at the β₂-AR and the 3-fold increased β₂-ARselectivity of (R,S)-5 was an unexpected result. One possibleexplanation of these results is that the naphthyl moiety of (R,S)-5 doesnot interact with the site defined by Y308 and H296 and is directedtowards and binds to another site on the β₂-AR. This interaction alsoconveys or participates in subtype selectivity as well as increasedbinding affinity and agonist activity. Since the previous models ofβ₂-AR selectivity only employed (R,R)-isomers, it is possible that thissite has been overlooked.

Another explanation of the data is suggested by the “rockingtetrahedron” chiral recognition mechanism proposed by Sokolov andZefirov (Doklady Akademii Nauk SSSR 319: 1382-1383, 1991). In thisapproach to molecular chiral recognition, the enantiomeric ligands aresecured to a chiral selector by two binding interactions. Theinteractions must be non-equivalent and directional so that only oneorientation is possible. The tethered enantiomers still haveconformational mobility and the remaining moieties on the chiral centerwill sweep out overlapping but not identical steric volumes. Where andto what extent the chiral selector interacts with these steric volumes,determines the enantioselectivity of the process. If the chirality ofthe chiral selector places the interaction perpendicular to the plane ofthe ligand, no enantioselectivity is observed. As a deviation from theperpendicular increases, so does the enantioselectivity relative to theR or S configuration.

With (R,R)-5 and (R,S)-5, the interactions with the first binding areaand the steric restricted site of the CoMFA model are two non-equivalentand directional interactions that place the remaining constituents onthe second chiral center in the same, albeit mirror image, orientationrelative to the second binding area. As discussed above, theinteractions of the 1-napthyl moieties of compound 5 with Y308 and H296are believed to be the source of the observed β₂-AR selectivity. If the1-naphthyl rings sweep out overlapping but not identical steric volumes,then the observed K_(i)β₂ values and subtype selectivity indicate thefollowing: 1) the K_(i)β₂-AR values represent the sum total of theπ-hydrogen bond and π-π interactions between the 1-naphthyl moieties andY308 and H296, as well as additional non-β₂-AR specific interactionswith other residues such as W109 and N312; 2) the steric volume sweptout by (R,S)-5 increases the probability of interactions of Y308 andH296 with the π cloud of the naphtyl moiety relative to the (R,R)-5; and3) the steric volume swept out by (R,R)-5 increases the probability ofinteractions with non-β₂-AR specific sites relative to (R,S)-5.

The effect of the configuration at the second chiral center andconformational-based chiral selectivity is also illustrated by theaffinities and subtype selectivities of (R,R)-3, (R,S)-3 and (R)-7,Table 1. The inversion of the chirality at the second chiral carbon fromR to S, reduced the K_(i)β₂ value of the (R,S)-3/β₂-AR complex relativeto the (R,R)-3/β₂-AR complex by ˜3-fold while there was no significantdifference between their K_(i)β₁ values. The increased subtypeselectivity observed for (R,R)-3 relative to (R,S)-3, 9 versus 4,respectively, essentially reflects the differences in K_(i)β₂ values,which could be a reflection of increased conformational energy requiredto bring the aromatic portion of the aminoalkyl chain into contact withthe electropositive and electronegative regions that comprise the secondbinding area or a decrease in the probability that this interactionwould occur.

The removal of the methyl moiety on the second chiral center, andthereby the chirality at this site ((R)-7), had a similar effect asinverting the chirality at this site from R to S. The K_(i)β₂ values for(R)-7 was 32% higher than (R,S)-3 and there was no difference in theβ₂-AR selectivity, Table 1. These results suggest that for compound 3,the primary effect of the R configuration at the second chiral site wasto direct the aminoalkyl chain towards the second binding area whichincreased the probability of interacting with this site and reduces theconformational energy required to achieve this interaction.

A difference between compounds 3 and 5 is the steric areas swept out bythe aromatic substituents. In the case of compound 3, the phenyl ringproduces a smaller, more linear area, while with compound 5, the1-naphthyl ring system produces a relatively larger and broader area.These differences can be used to guide the synthesis of additionalderivatives.

In an example, (R,R)-2 and (R,S)-5 are chosen as possible candidates forthe development of a new selective β₂-AR agonist. These compounds mayhave increased and extended systemic exposures relative to thecommercially available rac-1 due to changes in molecular hydrophobicity,metabolic profile and transporter interactions.

The present example provides a pharmacophore model which may be used asa structural guide for the design of new compounds with β₂-ARselectivity which can be tested for use in the treatment of a desiredcondition, including congestive heart failure.

Example 9 Pharmacokinetic studies of (R,R)-fenoterol,(R,R)-methoxyfenoterol and (R,S)-naphtylfenoterol

This example demonstrates the plasma concentrations of (R,R)-Fenoterol,(R,R)-Methoxyfenoterol and (R,S)-Naphtylfenoterol administered as anintravenous (IV) bolus to male Sprague-Dawley rats.

(R,R)-Fenoterol, (R,R)-Methoxyfenoterol and (R,S)-Naphtylfenoterol wereadministered to jugular vein cannulated (JVC) rats at a single dosage of5 mg/ml intravenously (see Table 3). Dose calculations (mg/kg) werebased on the individual body weight measured on the day of treatment.Study duration for pharmacokinetic studies was 6 hours. Plasma sampleswere collected over six hours at the following nine timepoints: prior toadministration of the desired dose; 5.00-5.30 minutes after dose;15.00-16.30 minutes after dose; 30.00-33.00 minutes after dose; 60-65minutes after dose; 120-125 minutes after dose; 240-245 minutes afterdose; 300-305 minutes after dose; and 360-365 minutes after dose. Urinewas collected for 0-6 hours and 6 to 24 hours from 3 rats in eachtreatment group.

TABLE 3 Study conditions for measuring plasma concentrations of (R,R)-fenoterol, (R,R)-methoxyfenoterol and (R,S)-naphtylfenoterol. Dose DoseNo. of Rats for level Concentration No. of plasma Compound: (mg/kg):(mg/ml): Rats: analysis: (R,R)-fenoterol 5 2.5 6 5 (R,R)- 5 2.5 6 5methoxyfenoterol (R,S)- 5 2.5 6 2 naphtylfenoterol

Pharmacokinetic parameters for (R,R)-fenoterol, (R,R)-methoxyfenoteroland (R,S)-naphtylfenoterol after intravenous administration to rats (5mg/kg) were analyzed according to a two-compartment open model (seeTable 4). A drug that follows the pharmacokinetics of a two-compartmentmodel does not equilibrate rapidly throughout the body, as is assumedfor a one-compartment model. In the two-compartment model, the drugdistributes into two compartments, the central compartment and thetissue, or peripheral compartment. The central compartment representsthe blood, extracellular fluid, and highly perfused tissues. The drugdistributes rapidly and uniformly in the central compartment. A secondcompartment, known as the tissue or peripheral compartment, containstissues in which the drug equilibrates more slowly. Drug transferbetween the two compartments is assumed to take place by first-orderprocesses.

The following abbreviations are utilized in Table 4 below: alpha—macrorate constant associated with the distribution phase; beta—macro rateconstant associated with the elimination phase; A, B—zero time interceptassociated with the alpha phase and beta phase, respectively; AUC—areaunder the curve; T1/2 (K10)—half-life associated with the rate constantK10; K10—elimination rate—rate at which the drug leaves the system fromthe central compartment; K12—rate at which drug enters tissuecompartment from the central compartment; K21—rate at which drug enterscentral compartment from tissue compartment; V1—volume of distributionof the central compartment; V2—volume of distribution of the tissuecompartment; Vss—volume of distribution at steady state; andCl—clearance.

TABLE 4 Pharmacokinetic parameters for (R,R)-fenoterol, (R,R)-methoxyfenoterol and (R,S)-naphtylfenoterol after intravenousadministration to rats (5 mg/kg). (R,S)- (R,R)- (R,R)- naphtyl-fenoterol methoxy- fenoterol (n = 2) fenoterol (n = 5) Weight 306 ± (n =5) Weight 297 ± Parameter Units 11 Weight 296 ± 8 10 Two-compartmentopen model A μg/ml 1.6300 4.6437 4.0365 Alpha 1/min 0.0710 0.1982 0.1764B μg/ml 0.0577 0.3900 0.4372 Beta 1/min 0.0086 0.0054 0.0046 AUC min *μg/ 29.6861 96.1011 116.88 ml T_(1/2) (K10) min 12.19 13.23 18.11 K101/min 0.0568 0.0524 0.0383 K12 1/min 0.0119 0.1309 0.1213 K21 1/min0.0107 0.0203 0.0214 V1 ml 906.5 294.01 330.83 V2 ml 1005.20 1895.001872.92 Vss ml 1911.70 2189.02 2203.75 Cl ml/min 51.54 15.40 12.66

Tables 5-7 and FIG. 8 illustrate the individual plasma concentrations of(R,R)-fenoterol, (R,R)-methoxyfenoterol and (R,S)-naphtylfenoterol afterIV administration to rats (5 mg/kg). The average concentration of(R,R)-fenoterol in plasma was dramatically lower (1.34 μg/ml) fiveminutes after IV administration to rats (5 mg/kg) compared to either theaverage concentration of (R,R)-methoxyfenoterol (2.12 μg/ml) or(R,S)-naphtylfenoterol (2.11 μg/ml).

TABLE 5 Individual plasma concentrations of (R,R)-fenoterol afterintravenous administration (5 mg/kg). Concentration (ug/ml) Time (min)Rat# 01 Rat #02 Average 5 1.34 1.34 15 0.36 0.36 30 0.17 0.50 0.34 600.05 0.05 0.05 120 0.03 0.01 0.02 240 0.0003 0.02 0.01 300 0.005 0.005360 0.08 0.03 0.06

TABLE 6 Individual plasma concentrations of (R,R)-methoxyfenoterol afterintravenous administration (5 mg/kg). Concentration (ug/ml) Rat# Rat#Rat# Rat# Rat# Time (min) 13 14 15 16 18 Average 5 1.94 2.14 2.51 1.892.12 15 0.48 0.54 0.62 0.67 0.56 0.58 30 0.31 0.40 0.46 0.48 0.38 0.4160 0.23 0.25 0.24 0.33 0.25 0.26 120 0.14 0.16 0.18 0.21 0.14 0.17 2400.09 0.12 0.17 0.14 0.08 0.12 300 0.06 0.07 0.07 0.09 0.07 0.07 360 0.050.06 0.05 0.08 0.04 0.06

TABLE 7 Individual plasma concentrations of (R,S)-naphtylfenoterol afterintravenous administration (5 mg/kg). Concentration (ug/ml) Rat# Rat#Rat# Rat# Rat# Time (min) 25 26 27 28 29 Average 5 2.52 2.16 1.64 2.102.11 15 0.85 0.78 0.50 0.60 0.68 30 0.49 0.54 0.34 0.33 0.43 60 0.360.42 0.37 0.29 0.24 0.34 120 0.25 0.29 0.26 0.22 0.18 0.24 240 0.11 0.110.13 0.13 0.11 0.12 300 0.10 0.11 0.12 0.11 0.10 0.11 360 0.08 0.08 0.110.10 0.09 0.09

The data demonstrate that the two derivatives, (R,R)-methoxyfenoteroland (R,S)-naphtylfenoterol, have a significantly higher systemicexposure (AUC) and longer clearance compared to (R,R)-fenoterol whichmay produce a longer acting drug. It is suggested that the longerclearance time may be the result of inhibiting glucuronidation.

Example 10 Treatment of Cardiac Disorders with (R,R)-Fenoterol orFenoterol Analogues

Based upon the teaching disclosed herein, a cardiac disorder such ascongestive heart failure is treated by administering a therapeuticeffective dose of (R,R)-fenoterol or one or more of the fenoterolanalogues disclosed above (see Sections III and IV). In an example, asubject who has been diagnosed with congestive heart failure isidentified. Following subject selection, a therapeutic effective dose of(R,R)-fenoterol or the respective fenoterol analogue is administered tothe subject. For example, a therapeutic effective dose of the(R,R)-fenoterol analogue including a OCH₃ group or a naphthyl derivativeis administered to the subject. In a further example, a therapeuticeffective dose of the (R,S)-fenoterol analogue including a napthylderivative is administered to the subject. The fenoterol analogue isprepared and purified as described in Section III.B and Example 5. Theamount of (R,R)-fenoterol or fenoterol analogue or a pharmaceuticalcomposition thereof administered to reduce, inhibit, and/or treatcongestive heart failure depends on the subject being treated, theseverity of the disorder, and the manner of administration of thetherapeutic composition (see Section V). Ideally, a therapeuticallyeffective amount of an agent is the amount sufficient to prevent,reduce, and/or inhibit, and/or treat the cardiac disorder (e.g.,congestive heart failure) in a subject without causing a substantialcytotoxic effect in the subject.

In an example, (R,R)-fenoterol, a disclosed fenoterol analogue (such asan (R,R)-fenoterol analogue including a OCH₃ group or a naphthylderivative or an (R,S)-fenoterol analogue including a napthylderivative) or pharmaceutical composition is provided at a dosage rangefrom about 0.001 to about 10 mg/kg body weight orally in single ordivided doses. In particular examples, the dosage range is from about0.005 to about 5 mg/kg body weight orally in single or divided doses(assuming an average body weight of approximately 70 kg; values adjustedaccordingly for persons weighing more or less than average).

In certain examples, a disclosed fenoterol compound or pharmaceuticalcomposition is provided by oral administration in the form of a tabletcontaining from about 1.0 to about 50 mg of the active ingredient,particularly about 2.0 mg to about 10 mg, more particularly about 2.5mg, about 5 mg, or about 10 mg of the active ingredient for thesymptomatic adjustment of the dosage to the subject being treated. Inone exemplary oral dosage regimen, a tablet containing from about 1 mgto about 50 mg active ingredient is administered two to four times aday. For example, a tablet containing about 1 mg to about 10 mg activeingredient is administered two times a day.

Example 11 Treatment of Pulmonary Disorders with Fenoterol Analogues

According to the teachings herein, a pulmonary disorder such as asthmaor chronic obstructive pulmonary disease is treated by administering atherapeutic effective dose of the fenoterol analogues disclosed above(see Sections III-V). In an example, a subject who has been diagnosedwith or displays one of the symptoms associated with asthma or chronicobstructive pulmonary disease is identified. Following subjectselection, a therapeutic effective dose of the desired fenoterolanalogue is administered to the subject. For example, a therapeuticeffective dose of the (R,R)-fenoterol analogue including a OCH₃ group ora naphthyl derivative is administered to the subject. In a furtherexample, a therapeutic effective dose of the (R,S)-fenoterol analogueincluding a napthyl derivative is administered to the subject. Thefenoterol analogue is prepared and purified as described in SectionIII.B and Example 5. The amount of the fenoterol analogue administeredto prevent, reduce, inhibit, and/or treat the pulmonary disorder dependson the subject being treated, the severity of the disorder, and themanner of administration of the therapeutic composition. Ideally, atherapeutically effective amount of an agent will be the amountsufficient to prevent, reduce, and/or inhibit, and/or treat thepulmonary disorder in a subject without causing a substantial cytotoxiceffect in the subject.

In an example, (R,R)-fenoterol, a disclosed fenoterol analogue (such asan (R,R)-fenoterol analogue including a OCH₃ group or a naphthylderivative or an (R,S)-fenoterol analogue including a napthylderivative) or pharmaceutical composition is provided at a dosage rangefrom about 0.001 to about 10 mg/kg body weight orally in single ordivided doses. In particular examples, the dosage range is from about0.005 to about 5 mg/kg body weight orally in single or divided doses(assuming an average body weight of approximately 70 kg; values adjustedaccordingly for persons weighing more or less than average).

In certain examples, a disclosed fenoterol compound or pharmaceuticalcomposition is provided by oral administration in the form of a tabletcontaining from about 1.0 to about 50 mg of the active ingredient,particularly about 2.0 mg to about 10 mg, more particularly about 2.5mg, about 5 mg, or about 10 mg of the active ingredient for thesymptomatic adjustment of the dosage to the subject being treated. Inone exemplary oral dosage regimen, a tablet containing from about 1 mgto about 50 mg active ingredient is administered two to four times aday. For example, a tablet containing about 1 mg to about 10 mg activeingredient is administered two times a day.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A compound having the formula:

wherein R₁-R₃ independently are hydrogen, acyl, alkoxy carbonyl or acombination thereof; R₄ is H or lower alkyl; R₅ is lower alkyl,

wherein Y¹, Y² and Y³ independently are hydrogen, lower —OR₆ and —NR₇R₈;wherein X and Y independently are selected from H, —OR₆ and —NR₇R₈; R₆is H or lower alkyl; and R₇ and R₈ independently are hydrogen, loweralkyl, alkoxy carbonyl, acyl or amino carbonyl.
 2. The compound of claim1, wherein R₄ is lower alkyl and R₅ is

wherein X is H, —OR₆ or —NR₇R₈; R₆ is lower alkyl; and R₇ and R₈independently are hydrogen or lower alkyl.
 3. The compound of claim 2,wherein R₄ is selected from methyl, ethyl, n-propyl, and isopropyl. 4.The compound of claim 3, wherein R₄ is methyl.
 5. The compound of claim4, wherein R₅ has the formula

wherein X is H, —OR₆ or —NR₇R₈.
 6. The compound of claim 5, wherein R₆is methyl.
 7. The compound of claim 6, wherein the compound is a(R,R′)-compound.
 8. The compound of claim 1, wherein the compound is a(R,S′)-compound.
 9. A pharmaceutical composition comprising the compoundof claim 1 and at least one pharmaceutically acceptable carrier.
 10. Amethod for treating a pulmonary or cardiovascular disorder in a subject,comprising: administering a therapeutically effective amount of thecompound of claim 2 to the subject with the pulmonary or cardiovasculardisorder.
 11. A method for treating a pulmonary or cardiovasculardisorder in a subject, comprising: administering a therapeuticallyeffective amount of the pharmaceutical composition of claim 9 to thesubject with the pulmonary or cardiovascular disorder.
 12. A method fortreating a pulmonary or cardiovascular disorder in a subject,comprising: administering a therapeutically effective amount of acompound having the formula:

wherein R₁-R₃ independently are hydrogen, acyl, alkoxy carbonyl or acombination thereof; R₄ is H or lower alkyl; R5 is

to the subject with the pulmonary or cardiovascular disorder and whereinthe compound is optically active.
 13. The method of claim 12, whereinthe compound is a (R,R′)-compound.
 14. The method of claim 12, whereinthe compound is a (R,S′)-compound.
 15. The method of claim 12, whereinthe disorder is a cardiovascular disorder.
 16. The method of claim 15,wherein the cardiovascular disorder is congestive heart failure.
 17. Themethod of claim 12, wherein the disorder is a pulmonary disorder. 18.The method of claim 17, wherein the pulmonary disorder is asthma orchronic pulmonary obstructive disease.
 19. A method for treating acardiovascular disorder in a subject, comprising: administering aneffective amount of a substantially optically pure (R,R)-fenoterol tothe subject with the cardiovascular disorder.
 20. The method of claim19, wherein the cardiovascular disorder is congestive heart failure.